Fusion polypeptides comprising mucin-domain polypeptide linkers

ABSTRACT

The invention provides fusion proteins having improved bioactivity comprising a first polypeptide fusion partner and a second polypeptide fusion partner wherein the first fusion partner is linked to the second fusion partner by a mucin-domain polypeptide linker and wherein the bioactivity of the fusion protein of the invention is improved as compared to fusion of the first polypeptide fusion partner and the second polypeptide fusion partner in the absence of the mucin-domain polypeptide linker. Mucin-domain polypeptide linkers comprise a mucin domain that is rich in potential glycosylation sites, and has a high content of serine and/or threonine and proline, which can represent greater than 40% of the amino acids within the mucin domain and further comprise at least about 60% of its mass due to the glycans.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/911,834, filed Jun. 6, 2013 which claims the benefit of U.S.Provisional Application Nos. 61/657,285, filed Jun. 8, 2012; 61/778,812,filed Mar. 13, 2013; 61/657,264, filed on Jun. 8, 2012; 61/778,575,filed Mar. 13, 2013; 61/657,378, filed Jun. 8, 2012 and 61/723,081,filed Nov. 6, 2012. The entire teachings of the above applications areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 31, 2013, isnamed 4000.3060WO_SL.txt and is 38,144 bytes in size.

BACKGROUND OF THE INVENTION

The construction of a fusion protein involves the linking of twoproteins or domains of proteins by a peptide linker. Selection of anappropriate linker sequence is important, as it can affect the functionand physical properties of the resulting fusion protein. Often flexibleand hydrophilic linkers are chosen so as to not overly constrain andthereby disturb the functions of the domains. The linkers can be used tocontrol the distance and the orientation of the domains. Fusion of abioactive protein or peptide often results in loss of bioactivity,likely due to steric interference of the fusion partner. Additionally,in the case of Fc fusions, due to their dimeric nature, interference canalso occur between the two copies of the heterologous protein.

Mucin proteins and mucin-domains of proteins contain a high degree ofglycosylation which structurally allows mucin proteins and otherpolypeptides comprising mucin domains to behave as stiffened randomcoils. The present invention is based, in part, on the discovery thatthis stiffened random coiled structure in combination with thehydrophilic branched hydrophilic carbohydrates that make up the heavilyglycosylated mucin domains is particularly useful as a linker in afusion protein. The rod-like nature of the mucin domains can rigidlyseparate the bioactive protein from the fusion partner, and thereby beless susceptible to loss in activity. In the case of Fc fusions, therigid projection away from the Fc will result in greater separationbetween each copy of the protein of interest, also enabling for largerfusion proteins to be expressed as Fc fusions. Also because of the highlevel of glycosylation, addition of a mucin domain has the potential tomodify the physicochemical properties of a protein such as charge,solubility and viscoelastic properties of concentrated solutions of theactive protein.

SUMMARY OF THE INVENTION

The present invention provides fusion proteins having improvedbioactivity comprising a first polypeptide fusion partner and a secondpolypeptide fusion partner wherein the first fusion partner is linked tothe second fusion partner by a mucin-domain polypeptide linker andwherein the bioactivity of the fusion protein of the invention isimproved as compared to fusion of the first polypeptide fusion partnerand the second polypeptide fusion partner in the absence of themucin-domain polypeptide linker. Mucin-domain polypeptide linkerscomprise a mucin domain that is rich in potential glycosylation sites,and has a high content of serine and/or threonine and proline, which canrepresent greater than 40% of the amino acids within the mucin domainand further comprise at least about 60% of its mass due to the glycans.Mucin domains polypeptide linkers may comprise tandem amino acid repeatunits (also referred to herein as TR) that may vary in length from about8 amino acids to 150 amino acids per each tandem repeat unit. The numberof tandem repeat units may vary between 1 and 5 in a mucin-domainpolypeptide linker of the invention.

Mucin-domain polypeptide linkers are capable of rigidly separating thefirst and second polypeptide fusion partners thereby decreasing thepossibility that one fusion partner will interfere with the biologicalactivity of the other fusion partner. The high level of glycosylation ofthe mucin-polypeptide linkers provides protection of proteolysis andpotentially increases the solubility of the one or both of the fusionpartners. When the fusion protein is a human therapeutic, themucin-domain linker may be derived from fully human sequences and thehigh level of glycosylation also reduces the risk if immunogenicity in ahuman. The desired degree of separation between the fusion partners maybe customized to provide maximum activity of the fusion protein byvarying the number of tandem repeats comprising the mucin domainpolypeptide.

A mucin-domain polypeptide linker may be used alone or in combinationwith an additional flexible linker sequence, and may also comprise a tagfor purification. The mucin-domain polypeptide linker may also impartimproved properties (e.g. pharmacokinetic and/or physicochemicalproperties) on the fusion protein compared to the fusion protein thatdoes not comprise a mucin-domain polypeptide linker

Nucleic acids encoding the polypeptides and methods for making thepolypeptides are also provided. The fusion proteins of this inventioncan be made by transforming host cells with nucleic acid encoding thefusion, culturing the host cell and recovering the fusion from theculture, or alternatively by generating a nucleic acid constructencoding the fusion and producing the polypeptide by cell freesynthesis, which synthesis may include coupled transcription andtranslation reactions. Also provided are vectors and polynucleotidesencoding the fusion protein.

The fusion proteins may be purified and formulated in pharmacologicallyacceptable vehicles for administration to a patient. In one embodimentof the invention the fusion protein comprises at least one domain of animmunoglobulin, e.g. a variable region domain; a constant region domain;a single chain Fv fragment; etc. Such fusion proteins find use asimmunologically specific reagents; e.g. to increase the plasma half-lifeof a polypeptide of interest or to target the protein to a particularcell type.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Base constructs and constructs containing a mucin linker.RDB1800 is an IL-1Ra_Fc fusion protein, RDB1819 in analogous to RDB1800but contains an intervening mucin sequence between the IL-1Ra and Fcdomains. Similarly, RDB1601 is a gp130(D1-D3)Fc fusion protein, with theD1 through D3 domains of gp130 directly linked to an IgG1 Fc domain, andRDB1613 contains a mucin sequence between gp130(D1-D3) and the Fcdomains.

FIG. 2: Inhibition of IL1β signaling by IL-1Ra (

), RDB1800 (

) and RDB1819 (

) in the HEK-Blue™ cell assay. Estimated values of IC₅₀ are reported inthe top right corner of the figure.

FIG. 3: Gel filtration chromatogram of RDB1800 (light grey) and RDB1819(dashed, dark grey) and molecular size standards (solid, dark grey).Molecular weights of the standards are listed above their eluting peaks.

FIG. 4: The inhibitory effects of a single 20 mg/kg injection of RDB1800and RDB1819 in the mouse CAIA model of inflammation. The black arrowsindicate the days of injection with the monoclonal antibody cocktail(mAb) and with LPS and treatment molecule. The % reduction in paw edemawas calculated for each compound compared to saline control. A group often mice were used for each treatment.

FIG. 5: Inhibition of IL-6-mediated differentiation of M1 cells RDB1601(

) and RDB1613 (

). Estimated values of IC₅₀ are reported in the table.

FIG. 6: Inhibition of IL-6 mediated differentiation of M1 cells byRDB1542 and RDB1562.

FIG. 7: IL-1β-dependent signaling in HEK-Blue™ IL-1β cells by RDB1840and RDB1841.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

DEFINITIONS

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The terms “polypeptide”, “peptide”, and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified, forexample, by disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component.

As used herein the term “amino acid” refers to either natural and/orunnatural or synthetic amino acids, including but not limited to glycineand both the D or L optical isomers, and amino acid analogs andpeptidomimetics. Standard single or three letter codes are used todesignate amino acids.

The term “non-naturally occurring,” as applied to sequences and as usedherein, means polypeptide or polynucleotide sequences that do not have acounterpart to, are not complementary to, or do not have a high degreeof homology with a wild-type or naturally-occurring sequence found in amammal. For example, a non-naturally occurring polypeptide may share nomore than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acidsequence identity as compared to a natural sequence when suitablyaligned.

The terms “glycosylation” and “glycosylated” are used interchangeablyherein to mean the carbohydrate portion of a protein or the process bywhich sugars are post-translationally attached to proteins during theirproduction in cells to form glyco-proteins. O-linked glycosylation ofproteins is a post-translational event and refers to the attachment ofglycans to serine and threonine and, to a lesser extent tohydroxyproline and hydroxylysine.

A “fragment” is a truncated form of a native active protein that retainsat least a portion of the therapeutic and/or biological activity. A“variant” is a protein with sequence homology to the native activeprotein that retains at least a portion of the therapeutic and/orbiological activity of the active protein. For example, a variantprotein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% amino acid sequence identity with the reference active protein.As used herein, the term “active protein moiety” includes proteinsmodified deliberately, as for example, by site directed mutagenesis,insertions, or accidentally through mutations.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient for the subject vectors. Host cells includeprogeny of a single host cell. The progeny may not necessarily becompletely identical (in morphology or in genomic of total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation. A host cell includes cells transfected in vivo witha vector of this invention.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. As is apparent to those of skill in the art,a non-naturally occurring polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, does not require “isolation” todistinguish it from its naturally occurring counterpart. In addition, a“concentrated”, “separated” or “diluted” polynucleotide, peptide,polypeptide, protein, antibody, or fragments thereof, is distinguishablefrom its naturally occurring counterpart in that the concentration ornumber of molecules per volume is generally greater than that of itsnaturally occurring counterpart. In general, a polypeptide made byrecombinant means and expressed in a host cell is considered to be“isolated.”

An “isolated” polynucleotide or polypeptide-encoding nucleic acid orother polypeptide-encoding nucleic acid is a nucleic acid molecule thatis identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal or extra-chromosomal location differentfrom that of natural cells.

“Conjugated”, “linked,” “fused,” and “fusion” are used interchangeablyherein. These terms refer to the joining together of two more chemicalelements or components, by whatever means including chemical conjugationor recombinant means. For example, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and in reading phase or in-frame. An“in-frame fusion” refers to the joining of two or more open readingframes (ORFs) to form a continuous longer ORF, in a manner thatmaintains the correct reading frame of the original ORFs. Thus, theresulting recombinant fusion protein is a single protein containing twoor more segments that correspond to polypeptides encoded by the originalORFs (which segments are not normally so joined in nature).

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminus direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide. A“partial sequence” is a linear sequence of part of a polypeptide that isknown to comprise additional residues in one or both directions.

“Heterologous” means derived from a genotypically distinct entity fromthe rest of the entity to which it is being compared. For example, aglycine rich sequence removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous glycine rich sequence. The term “heterologous” asapplied to a polynucleotide, a polypeptide, means that thepolynucleotide or polypeptide is derived from a genotypically distinctentity from that of the rest of the entity to which it is beingcompared.

The terms “polynucleotides”, “nucleic acids”, “nucleotides” and“oligonucleotides” are used interchangeably. They refer to a polymericform of nucleotides of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a

“Recombinant” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of in vitrocloning, restriction and/or ligation steps, and other procedures thatresult in a construct that can potentially be expressed in a host cell.

The terms “gene” or “gene fragment” are used interchangeably herein.They refer to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated. A gene or gene fragment may be genomic orcDNA, as long as the polynucleotide contains at least one open readingframe, which may cover the entire coding region or a segment thereof. A“fusion gene” is a gene composed of at least two heterologouspolynucleotides that are linked together.

“Homology” or “homologous” refers to sequence similarity orinterchangeability between two or more polynucleotide sequences or twoor more polypeptide sequences. When using a program such as BestFit todetermine sequence identity, similarity or homology between twodifferent amino acid sequences, the default settings may be used, or anappropriate scoring matrix, such as blosum45 or blosum80, may beselected to optimize identity, similarity or homology scores.Preferably, polynucleotides that are homologous are those whichhybridize under stringent conditions as defined herein and have at least70%, preferably at least 80%, more preferably at least 90%, morepreferably 95%, more preferably 97%, more preferably 98%, and even morepreferably 99% sequence identity to those sequences.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Generally,stringency of hybridization is expressed, in part, with reference to thetemperature and salt concentration under which the wash step is carriedout. Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short polynucleotides (e.g., 10to 50 nucleotides) and at least about 60° C. for long polynucleotides(e.g., greater than 50 nucleotides) for example, “stringent conditions”can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C.,and three washes for 15 min each in 0.1×SSC/1% SDS at 60 to 65° C.Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C.may be used. SSC concentration may be varied from about 0.1 to 2×SSC,with SDS being present at about 0.1%. Such wash temperatures aretypically selected to be about 5° C. to 20° C. lower than the thermalmelting point for the specific sequence at a defined ionic strength andpH. The Tm is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. An equation for calculating Tm and conditions for nucleic acidhybridization are well known and can be found in Sambrook, J. et al.(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Vol. 1-3,Cold Spring Harbor Press, Plainview N.Y.; specifically see Volume 2 andChapter 9. Typically, blocking reagents are used to block non-specifichybridization. Such blocking reagents include, for instance, sheared anddenatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, suchas formamide at a concentration of about 35-50% v/v, may also be usedunder particular circumstances, such as for RNA:DNA hybridizations.Useful variations on these wash conditions will be readily apparent tothose of ordinary skill in the art.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences. Percent identity may bemeasured over the length of an entire defined polynucleotide sequence,for example, as defined by a particular SEQ ID number, or may bemeasured over a shorter length, for example, over the length of afragment taken from a larger, defined polynucleotide sequence, forinstance, a fragment of at least 45, at least 60, at least 90, at least120, at least 150, at least 210 or at least 450 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the Tables, Figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Percent (%) amino acid sequence identity,” with respect to thepolypeptide sequences identified herein, is defined as the percentage ofamino acid residues in a query sequence that are identical with theamino acid residues of a second, reference polypeptide sequence or aportion thereof, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Percentidentity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the Tables, Figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

A “vector” is a nucleic acid molecule, preferably self-replicating in anappropriate host, which transfers an inserted nucleic acid molecule intoand/or between host cells. The term includes vectors that functionprimarily for insertion of DNA or RNA into a cell, replication ofvectors that function primarily for the replication of DNA or RNA, andexpression vectors that function for transcription and/or translation ofthe DNA or RNA. Also included are vectors that provide more than one ofthe above functions. An “expression vector” is a polynucleotide which,when introduced into an appropriate host cell, can be transcribed andtranslated into a polypeptide(s). An “expression system” usuallyconnotes a suitable host cell comprised of an expression vector that canfunction to yield a desired expression product.

“Degradation resistance,” as applied to a polypeptide, refers to theability of the polypeptides to withstand degradation in blood orcomponents thereof, which typically involves proteases in the serum orplasma, or within a formulation intended as a storage or deliveryvehicle for a protein. The degradation resistance can be measured bycombining the protein with human (or mouse, rat, monkey, as appropriate)blood, serum, plasma, or a formulation, typically for a range of days(e.g., 0.25, 0.5, 1, 2, 4, 8, 16 days), at specified temperatures suchas −80° C., −20° C., 0° C., 4° C., 25° C., and 37° C. The intact proteinin the samples is then measured using standard protein quantitationtechniques. The time point where 50% of the protein is degraded is the“degradation half-life” of the protein.

The term “half-life” typically refers to the time required for theplasma concentration of a drug to be reduced by one-half. The terms“half-life”, “t_(1/2)”, “elimination half-life” and “circulatinghalf-life” are used interchangeably herein.

“Apparent Molecular Weight” is a term referring to a measure of therelative increase or decrease in apparent molecular weight exhibited bya particular amino acid sequence. The apparent molecular weight isdetermined using size exclusion chromatography (SEC) and similar methodscompared to globular protein standards and is measured in “apparent kD”units.

The “hydrodynamic radius” is the apparent radius (R_(h) in nm) of amolecule in a solution calculated from diffusional properties. The“hydrodynamic radius” of a protein affects its rate of diffusion inaqueous solution as well as its ability to migrate in gels ofmacromolecules. The hydrodynamic radius of a protein is influenced byits molecular weight as well as by its structure, including shape andcompactness, and its hydration state. Methods for determining thehydrodynamic radius are well known in the art, such as by the use of DLSand size exclusion chromatography. Most proteins have globularstructure, which is the most compact three-dimensional structure aprotein can have with the smallest hydrodynamic radius. Some proteinsadopt a random and open, unstructured, or ‘linear’ conformation and as aresult have a much larger hydrodynamic radius compared to typicalglobular proteins of similar molecular weight.

“Physiological conditions” refer to a set of conditions in a living hostas well as in vitro conditions, including temperature, saltconcentration, pH, that mimic those conditions of a living subject. Ahost of physiologically relevant conditions for use in in vitro assayshave been established. Generally, a physiological buffer contains aphysiological concentration of salt and is adjusted to a neutral pHranging from about 6.5 to about 7.8, and preferably from about 7.0 toabout 7.5. A variety of physiological buffers is listed in Sambrook etal. (1989). Physiologically relevant temperature ranges from about 25°C. to about 38° C., and preferably from about 35° C. to about 37° C.

“Controlled release agent”, “slow release agent”, “depot formulation” or“sustained release agent” are used interchangeably to refer to an agentcapable of extending the duration of release of a polypeptide of theinvention relative to the duration of release when the polypeptide isadministered in the absence of agent.

The term “antagonist”, as used herein, includes any molecule thatpartially or fully blocks, inhibits, or neutralizes a biologicalactivity of a native polypeptide disclosed herein. Methods foridentifying antagonists of a polypeptide may comprise contacting anative polypeptide with a candidate antagonist molecule and measuring adetectable change in one or more biological activities normallyassociated with the native polypeptide. In the context of the presentinvention, antagonists may include proteins, nucleic acids,carbohydrates, antibodies or any other molecules that decrease theeffect of an active protein.

The term “agonist” is used in the broadest sense and includes anymolecule that mimics a biological activity of a native polypeptidedisclosed herein. Suitable agonist molecules specifically includeagonist antibodies or antibody fragments, fragments or amino acidsequence variants of native polypeptides, peptides, small organicmolecules, etc. Methods for identifying agonists of a native polypeptidemay comprise contacting a native polypeptide with a candidate agonistmolecule and measuring a detectable change in one or more biologicalactivities normally associated with the native polypeptide.

“Activity” for the purposes herein refers to an action or effect of acomponent of a fusion protein consistent with that of the correspondingnative active protein, wherein “biological activity” or “bioactivity” asthose terms are used interchangeably herein refers to an in vitro or invivo biological function or effect, including but not limited toreceptor binding, antagonist activity, agonist activity, or a cellularor physiologic response.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” is used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the subject, notwithstanding that thesubject may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a subjectat risk of developing a particular disease, or to a subject reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect”, as used herein, refers to a physiologic effect,including but not limited to the cure, mitigation, amelioration, orprevention of disease in humans or other animals, or to otherwiseenhance physical or mental well-being of humans or animals, caused by afusion protein of the invention other than the ability to induce theproduction of an antibody against an antigenic epitope possessed by theactive protein. Determination of a therapeutically effective amount iswell within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose”, as used herein, refers to an amount of an activeprotein, either alone or as a part of a fusion protein composition, thatis capable of having any detectable, beneficial effect on any symptom,aspect, measured parameter or characteristics of a disease state orcondition when administered in one or repeated doses to a subject. Sucheffect need not be absolute to be beneficial.

The term “therapeutically effective dose regimen”, as used herein,refers to a schedule for consecutively administered doses of an activeprotein, either alone or as a part of a fusion protein composition,wherein the doses are given in therapeutically effective amounts toresult in sustained beneficial effect on any symptom, aspect, measuredparameter or characteristics of a disease state or condition.

Fusion Proteins

In various aspects the invention provides fusion proteins comprising afirst polypeptide fusion partner linked to a second polypeptide fusionpartner by a mucin-domain polypeptide linker. As used herein, the terms“fusion protein” or “fusion polypeptide” or grammatical equivalentsherein are meant to denote a protein composed of a plurality of proteincomponents, which are typically unjoined in their native state but arejoined by their respective amino and carboxyl termini through amucin-domain polypeptide linker of the invention. “Protein” in thiscontext includes proteins, polypeptides and peptides. Plurality in thiscontext means at least two, and preferred embodiments generally utilizea first and a second polypeptide fusion partner joined through amucin-domain polypeptide linker in accordance with the invention.

At least one or both of the first and second polypeptide fusion partnersare active proteins and/or therapeutic active proteins as that term isdefined herein. In one embodiment the therapeutic/biological activity ofat least one of the polypeptide fusion partners is improved when linkedto the other fusion partner via a mucin-domain polypeptide linker inaccordance with the invention as compared to the same polypeptide fusionpartners not linked via a mucin-domain polypeptide linker in accordancewith the invention.

Mucin proteins and mucin-domains of proteins contain a high degree ofglycosylation which structurally allows mucin proteins and otherpolypeptides comprising mucin domains to behave as stiffened randomcoils. As such, mucin domains are present in a variety ofmembrane-anchored adhesion molecules and receptors (including, but notlimited to LDL receptor, CD164, endosialin, fractalkine, the selectins,TIM (transmembrane Ig mucin) family proteins) where their function is toextend the ‘active’ domain away from the cell surface for optimalinteraction (Fong et al., J. Biol. Chem, 275 (6), (2000)). By analogy,the stiffened random coiled structure in combination with thehydrophilic branched hydrophilic carbohydrates that make up the heavilyglycosylated mucin domains can be particularly useful for providingcontrolling the separation between the two fusion partners as forcontrolling the length and rigidity of the separation between two fusionpartners.

Additionally, the hydrophilic branched hydrophilic carbohydrates thatmake up the heavily glycosylated mucin domains of the mucin-domainpolypeptide linker are desirable for increasing the hydrodynamic radiusof the fusion protein beyond what would be expected solely based on theadded molecular weight. Such increase in hydrodynamic imparts desirablequalities on the fusion protein such as, for example increasing theserum half-life of a therapeutic fusion protein.

The high level of glycosylation provided by the addition of a mucindomain polypeptide linker also has the potential to modify thephysicochemical properties of a protein such as charge, solubility andviscoelastic properties of concentrated solutions of the active protein.

One fusion protein design combines the binding region(s) of a firstpolypeptide fusion partner through a linker of the invention, to asecond polypeptide fusion partner that is all or a portion of animmunoglobulin. Generally, as the term is utilized in the specification,“immunoglobulin” or “immunoglobulin domain” is intended to include alltypes of immunoglobulins (IgG, IgM, IgA, IgE, IgD, etc.), from allsources (e.g., human, rodent, rabbit, cow, sheep, pig, dog, othermammal, chicken, turkey, emu, other avians, etc). Immunoglobulins fromhumans are preferred when the fusion proteins of the invention are usedfor treating humans.

In one embodiment, one or more immunoglobulin fusion partners comprisethe hinge and Fc regions of an immunoglobulin heavy chain. Typically, insuch N-terminal fusions the encoded fusion polypeptide will retain atleast functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of the fusionproteins. In some embodiments, the fusion proteins of the invention areassembled as monomers, or hetero- or homo-multimers, and particularly asdimers or tetramers as is known in the art. Although the presence of animmunoglobulin light chain is not required, an immunoglobulin lightchain might be present either covalently associated or directly fused tothe polypeptide.

In one embodiment, fusion partners comprise serum albumin or a domain ofserum albumin. Human serum albumin is preferred when the fusion proteinsof the invention are used for treating humans. In another embodiment,fusion partners comprise human transferrin.

Of particular interest are fusion proteins for which an increasebioactivity of the fusion protein is sought as compared to the samefusion of active proteins in the absence of a mucin-domain polypeptidelinker. Also of interest are fusion proteins for which an increase in apharmacokinetic parameter such as serum half-life, increased solubility,increased stability, or some other enhanced pharmaceutical property issought as compared to the same fusion of active proteins in the absenceof a mucin-domain polypeptide linker.

The activity of the fusion protein compositions of the invention,including functional characteristics or biologic and pharmacologicactivity and parameters that result, may be determined by any suitableassay known in the art for measuring the desired characteristic. Theactivity and structure of the fusion proteins may be measured by assaysdescribed herein, assays of the Examples, or by methods known in the artto ascertain the half-life, degree of solubility, structure andretention of biologic activity of the fusion proteins of the inventionas well as comparisons with active proteins that are not fusion proteinsof the invention. Quantitation of biologic activity (potency) assaysinclude, but are not limited by, in vitro binding assays (such as ELISA,surface plasmon resonance, thermal shift assays, NMR, sedimentation,scintillation proximity, FRET, fluorescence anisotropy), in vitrocell-based assays (such as reporter-gene, phosphorylation, celldifferentiation, cell growth or viability, enzyme complementarity, celllabeling), and in vivo pharmacological activities (including animalmodels of disease).

Fusion proteins of the invention may be produced via standard expressionmeans without the need for further conjugation and purification steps.Mucin-domain polypeptides linkers may be linked to one or both fusionpartners via either the N- or C-terminus of the fusion partner.Mucin-domain polypeptide liners are structurally less restrictive thanother fusion partners in that they are monomeric, non-globular proteinshaving reduced bulk and a lowered risk of impact on bioactivity.

When referring to the fusion protein, the term “linked” or “fused” or“fusion” is intended to indicate that the mucin-domain polypeptidelinkers and the polypeptide fusion partners are expressed as a singlepolypeptide in cells in a manner that allows for O-linked glycosylationof the mucin-domain polypeptide and maintains the activity of the activeprotein.

A fusion protein of the invention can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) Current Protocols in MolecularBiology, John Wiley & Sons, 1992). Many expression vectors arecommercially available to assist with fusion moieties and will bediscussed in more detail below.

Mucin-Domain Polypeptide Linkers

A “mucin-domain polypeptide linker” is defined herein as any proteincomprising a “mucin domain” capable of being linked to one or morefusion polypeptide partners. A mucin domain is rich in potentialglycosylation sites, and has a high content of serine and/or threonineand proline, which can represent greater than 40% of the amino acidswithin the mucin domain. A mucin domain is heavily glycosylated withpredominantly O-linked glycans. A mucin-domain polypeptide has at leastabout 60%, at least 70%, at least 80%, or at least 90% of its mass dueto the glycans. Mucin domains may comprise tandem amino acid repeatunits (also referred to herein as TR) that may vary in length from about8 amino acids to 150 amino acids per each tandem repeat unit. The numberof tandem repeat units may vary between 1 and 25 in a mucin-domainpolypeptide of the invention.

Mucin-domain polypeptide linkers of the invention include, but are notlimited to, all or a portion of a mucin protein. A “portion thereof” ismeant that the mucin polypeptide linker comprises at least one mucindomain of a mucin protein. Mucin proteins include any protein encodedfor by a MUC gene (e.g., MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B,MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19,MUC20, MUC21). The mucin domain of a mucin protein is typically flankedon either side by non-repeating amino acid regions. A mucin-domainpolypeptide may comprise all or a portion of a mucin protein (e.g.MUC20). A mucin-domain polypeptide may comprise all or a portion of amucin protein of a soluble mucin protein. Preferably the mucin-domainpolypeptide comprises the extracellular portion of a mucin protein.

A mucin domain polypeptide may also comprise all or a portion of aprotein comprising a mucin domain but that is not encoded by a MUC gene.Such naturally occurring proteins that are not encoded by a MUC gene butthat comprise mucin domains include, but are not limited to,membrane-anchored proteins such as transmembrane immunoglobulin andmucin domain (TIM) family proteins, fractalkine (neurotactin),P-selectin glycoprotein ligand 1 (PSGL-1, CD162), CD34, CD43(leukosialin, sialophorin), CD45, CD68, CD96, CD164, GlyCAM-1, MAdCAM,E-selectin, P-selectin, L-selectin, red blood cell glycophorins,glycocalicin, glycophorin, LDL-R, ZP3, endosialin, decay acceleratingfactor (daf, CD55), podocalyxin, endoglycan, alpha-dystroglycan,neurofascin, EMR1, EMR2, EMR3, EMR4, ETL and epiglycanin.

A mucin-domain polypeptide linker may also comprise a non-naturallyoccurring polypeptide having a mucin domain as that term is definedherein. In one embodiment, the mucin-domain polypeptide is designed denovo to comprise a mucin domain in accordance with the invention.

In one embodiment, the mucin-domain polypeptide linker is notglycosylated by α1,3, galactosyltransferase orβ1,6-acetylglucosaminyltransferase. In one embodiment, the fusionprotein does not bind an antibody specific for an αGal. In oneembodiment the fusion protein of the invention does not bind a Galα1,3Gal specific antibody.

In one embodiment a mucin domain polypeptide linker comprises domains oftandem amino acid repeats that are rich in Pro, Ser and Thr. In oneaspect of this embodiment, the number of tandem repeat units within amucin domain polypeptide linker of the invention is between 1 and 25.Preferably, the number of tandem repeat units within a mucin domainpolypeptide linker is between 2 and 20. More preferably, the number oftandem repeat units within a mucin domain polypeptide is at least about4. In a further aspect of this embodiment, the percentage of serineand/or threonine and proline residues within a mucin domain polypeptideof the invention is at least 10%. Preferably, the percentage of serineand/or threonine and proline residues within a mucin domain polypeptideof the invention is at least 20%. More preferably, the percentage ofserine and/or threonine and proline residues within a mucin domainpolypeptide of the invention is greater than 30%. In a final aspect ofthis embodiment, each tandem amino acid repeat unit within the mucindomain is comprised of at least 8 amino acids. Preferably, each unit iscomprised of at least 16 amino acids. More preferably, each unit iscomprised of at least 19 amino acids, and each unit may vary in lengthfrom about 19 amino acids to 150 amino.

In one embodiment the mucin-domain polypeptide comprises at least 32amino acids, comprising at least 40% Serine, Threonine, and Proline. Inone embodiment, a mucin-domain polypeptide in accordance with theinvention comprises at least 2, 4, 8, 10 or 12 tandem amino acidrepeating units of at least 8 amino acids in length per tandem repeatingunit. Preferred amino acid sequences of a tandem repeating unit include,but are not limited to those of Table I. The mucin-domain polypeptide,and/or nucleic acids encoding the mucin-domain polypeptide, may beconstructed using mucin-domain encoding sequences of proteins that areknown in the art and are publicly available through sources such asGenBank.

TABLE I Tandem Repeat (TR) Amino Acid Sequence Number of Accession Name(# of aa's) TR/MUC* Number⁺ Notes MUC1 PAPGSTAPPAHGVTSAPDTR (20) 21-125;41 and P15941 Multiple [SEQ ID NO: 5] 85 are variants of most commonMUC1 exist MUC2 ITTTTTVTPTPTPTGTQTPTTTP (23) 99 Q02817 Major TR; [SEQ IDNO: 6] alternative TR sequences exist MUC3 (A) ITTTETTSHDTPSFTSS (17) 20Q02505 Degenerate [SEQ ID NO: 7] TR sequence; long serine-rich andthreonine- rich sequence also exist MUC4 ATPLPVTDTSSASTGH (16) 145-395Q99102 Degenerate [SEQ ID NO: 8] TR sequence, long serine-rich andthreonine- rich sequence also exist MUC5AC TTSTTSAP (8)(46,17,34,58)^(∞) P98088 Consensus [SEQ ID NO: 9] sequence T-T-S-T-T-S-A-P (SEQ ID NO: 9) MUC5B ATGSTATPSSTPGTTHTPPVLTTTATTPT (29) (11, 11,17, 11, 23)^(∞) Q9HC84 Degenerate [SEQ ID NO: 10] TR sequence MUC6 PTSNA Q6W4X9 NA MUC7 TTAAPPTPSATTQAPPSSSAPPE (23) 5-6 Q8TAX7 Degenerate[SEQ ID NO: 11] TR sequence MUC11/12 EESTTVHSSPGATGTALFP (19) 28 Q9UKN1Consensus [SEQ ID NO: 12] sequence E-E-S-X- X-X-H-X- X-P-X-X- T-X-T-X-X-X-P (SEQ ID NO: 22) MUC13 PTS NA Q9H3R2 MUC14 PTS NA MUC15 PTS NAQ8N387 MUC16 PTS NA Q8WXI7 MUC17 SSSPTPAEGTSMPTSTYSEGRTPLTSMPVSTT 59-60Q685J3 Degenerate LVATSAISTLSTTPVDTSTPVTNSTEA (60) TR [SEQ ID NO: 13]sequence MUC19 PTS NA Q7Z5P9 Repeats of G-V-T-G- T-T-G-P- S-A (SEQ IDNO: 23) MUC20 SESSASSDGPHPVITPSRA (19) 11-12 Q8N307 [SEQ ID NO: 14]MUC21 ATNSESSTVSSGIST (15) 28 Q5SSG8 Degenerate [SEQ ID NO: 15] TRsequence MUC22 PTS NA E2RYF6 TIM-1 VPTTTT (6) 11 Q96D42 Degenerate [SEQID NO: 16] TR sequence TIM-4 PTS NA Q96H15 Fractalkine Mucin-like region(PTS) NA P78423 Macrosialin Mucin-like region (PTS) NA P34810 (CD68)CD96 PTS NA P40200 Endosialin Pro-rich region NA Q9HCU0 DAF Pro/Thr-richregion NA P08174 (CD55) Podocalyxin Thr-rich region NA O00592 EMR1Ser/Thr-rich region NA Q14246 PSGL-1 QTTQPAATEA (10) 12 Q14242Degenerate [SEQ ID NO: 17] TR sequence MUC8 and MUC9 are omitted; noreliable data PTS proline/serine/threonine rich sequence *approximate;TR number is reported as a range in most cases ⁺Uniprot number ^(∞)Thenumber n of TR is different in specific regions NA Not announced

Alternatively, the mucin-domain polypeptide linker is provided as avariant mucin-domain polypeptide having a mutation in thenaturally-occurring mucin-domain sequence of a wild type protein. Forexample, the variant mucin-domain polypeptide linker comprisesadditional O-linked glycosylation sites compared to the wild-typemucin-domain polypeptide. Alternatively, the variant mucin-domainpolypeptide comprises amino acid sequence mutations that result in anincreased number of serine, threonine or proline residues as compared toa wild type mucin-domain polypeptide Alternatively, the variantmucin-domain polypeptide sequences comprise added or subtracted chargedresidues, including but not limited to aspartatic acid, glutamic acid,lysine, histidine, and arginine, which change the pI or charge of themolecule at a particular pH.

Active Protein and Therapeutic Active Protein

As used herein an “active protein” when referring to a polypeptidefusion partner means a protein of biologic, therapeutic, prophylactic,or diagnostic interest or function and/or is capable of mediating abiological activity. A “therapeutic active protein” as that term is usedherein when referring to a polypeptide fusion partner, is a protein thatis capable of preventing or ameliorating a disease, disorder orconditions when administered to a subject. In a preferred embodiment, anactive protein or therapeutic active protein in accordance with theinvention refers to a fusion with serum albumin (or any fragmentthereof), an immunoglobulin molecule, an Fc domain of an immunoglobulin,or any fragment thereof.

An active protein of the invention can be a native, full-length protein,or can be a circularly permutated full-length protein, or can be afragment or a sequence variant of an active protein, or can be acircularly permutated fragment or circularly permutated sequence variantof an active protein, that retains at least a portion of the therapeuticactivity of the native active protein. In one embodiment, the activeproteins in accordance with the invention can be a recombinantpolypeptide with a sequence corresponding to a protein found in nature.

In another embodiment, the active proteins can be sequence variants,fragments, homologs, and mimetics of a natural sequence, or circularlypermutated sequence variants, fragments, homologs, and mimetics of anatural sequence that retain at least a portion of the biologicalactivity of the native active protein.

An active protein when referring to a polypeptide fusion partner of theinvention can itself be a fusion polypeptide. In one embodiment, saidactive protein that is itself a fusion polypeptide can be a fusionpolypeptide comprising two or more native, full-length proteins, or twoor more circularly permutated full-length proteins, or fragments orsequence variants of two or more active proteins, or circularlypermutated fragments or circularly permutated sequence variants of twoor more active proteins. In a further embodiment, said active proteinthat is itself a fusion polypeptide can be a fusion polypeptidecomprising one or more combinations of native full-length proteins,full-length circularly permutated proteins, fragments or sequencevariants of active proteins, circularly permutated fragments orcircularly permutated sequence variants of active proteins, that retainat least a portion of the biological activity of the native activeproteins. In another embodiment, the active protein that is itself afusion polypeptide in accordance with the invention can comprise arecombinant fusion polypeptide with sequences corresponding to proteinsfound in nature. In another embodiment, the active protein that isitself a fusion polypeptide can be sequence variants, fragments,homologs, and mimetics of natural sequences, or circularly permutatedsequence variants, fragments, homologs, and mimetics of naturalsequences that retain at least a portion of the biological activity ofthe native active proteins.

In non-limiting examples, the active protein can be a sequence thatexhibits at least about 80% sequence identity, or alternatively 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to the native activeprotein or a variant of a native active protein. Such proteins includebut are not limited to the following: bioactive peptides (such as GLP-1,exendin-4, oxytocin, opiate peptides), cytokines, growth factors,chemokines, lymphokines, ligands, receptors, hormones, enzymes,antibodies and antibody fragments, domain antibodies, nanobodies, singlechain antibodies, engineered antibody ‘alternative scaffolds’ such asDARPins, centyrins, adnectins, and growth factors Examples of receptorsinclude the extracellular domain of membrane associated receptors (suchas TNFRI, TNFR2, VEGF receptors, IL-1R1, IL-1RAcP, IL-4 receptor, hGHreceptor, CTLA-4, PD-1, IL-6Rα, FGF receptors, cytokine receptors oraccessory proteins), soluble receptors which have been cleaved fromtheir transmembrane domains, ‘dummy’ or ‘decoy’ receptors (such asIL-1RII, TNFRSF11B, DcR3), and any chemically or genetically modifiedsoluble receptors. Examples of enzymes include activated protein C,factor VII, collagenase; agalsidase-beta; dornase-alpha; alteplase;pegylated-asparaginase; asparaginase: and imiglucerase. Examples ofspecific polypeptides or proteins include, but are not limited togranulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), macrophage colony stimulating factor(M-CSF), colony stimulating factor (CSF), interferon beta (IFN-β),interferon gamma (FNTγ), interferon gamma inducing factor I (IGLF),transforming growth factor beta (TGF-β). RANTES (regulated uponactivation, normal T-cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1-α and MIP-1-β, Leishmania elongationinitiating factor (LEIF), platelet derived growth factor (PDGF), tumornecrosis factor (TNF), growth factors, e.g., epidermal growth factor(EGF), vascular endothelial growth factor (VEGF), fibroblast growthfactor, (FGF), nerve growth factor (NGF), brain derived neurotrophicfactor (BDNF), neurotrophin-2 (NT-2), neurotrophin-3 (NT-3),neurotrophin-4 (NT-4), neurotrophin-5 (NT-5), glial cell line-derivedneurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), TNF atype 11 receptor, erythropoietin (EPO), insulin and solubleglycoproteins e.g., gp120 and gp160 glycoproteins The gp120 glycoproteinis a human immunodeficiency virus (HIV) envelope protein, and the gp160glycoprotein is a known precursor to the gp120 glycoprotein.

In one embodiment, the biologically active polypeptide is GLP-1. Inanother embodiment, the biologically active polypeptide is nesiritide,human B-type natriuretic peptide (hBNP). In yet another embodiment, thebiologically active polypeptide is secretin, which is a peptide hormonecomposed of an amino acid sequence identical to the naturally occurringporcine secretin consisting of 27 amino acids. In one embodiment, thebiologically active polypeptide is enfuvirtide, a linear 36-amino acidsynthetic polypeptide which is an inhibitor of the fusion of HIV-1 withCD4+ cells. In one embodiment, the biologically active polypeptide isbivalirudin, a specific and reversible direct thrombin inhibitor.Antihemophilic Factor (AHF) may be selected as the active polypeptide.In another embodiment, erythropoietin is the biologically activepolypeptide. Erythropoietin is a 165 amino acid glycoproteinmanufactured by recombinant DNA technology and has the same biologicaleffects as endogenous erythropoietin. In still another embodiment, thebiologically active polypeptide is Reteplase. Reteplase is anon-glycosylated deletion mutein of tissue plasminogen activator (tPA),comprising the kringle 2 and the protease domains of human tPA.

In one preferred embodiment, the active polypeptide which is Anakirna, arecombinant, nonglycosylated form of the human interleukin-1 receptorantagonist (IL-IRa). In one case, Anakinra consists of 153 amino acidsand has a molecular weight of 17.3 kilodaltons. It may be produced byrecombinant DNA technology using an E. coli bacterial expression system.

Becaplermin may also be selected as the active polypeptide. Becaplerminis a recombinant human platelet-derived growth factor (rhPDGF-BB) fortopical administration. Becaplermin may be produced by recombinant DNAtechnology by insertion of the gene for the B chain of platelet derivedgrowth factor (PDGF) into the yeast strain Saccharomyces cerevisiae. Oneform of Becaplermin has a molecular weight of approximately 25 kD and isa homodimer composed of two identical polypeptide chains that are boundtogether by disulfide bonds. The active polypeptide may be Oprelvekin,which is a recombinant form of interleukin eleven (IL-11) that isproduced in Escherichia coli (E. coli) by recombinant DNA technology. Inone embodiment, the selected biologically active polypeptide has amolecular mass of approximately 19,000 daltons, and is non-glycosylated.The polypeptide is 177 amino acids in length and differs from the 178amino acid length of native IL-11 only in lacking the amino-terminalproline residue, which is known not to result in measurable differencesin bioactivity either in vitro or in vivo. Yet another embodimentprovides for a biologically active polypeptide which is Glucagon, apolypeptide hormone identical to human glucagon that increases bloodglucose and relaxes smooth muscles of the gastrointestinal tract.Glucagon may be synthesized in a special non-pathogenic laboratorystrain of E. coli bacteria that have been genetically altered by theaddition of the gene for glucagon. In a specific embodiment, glucagon isa single-chain polypeptide that contains 29 amino acid residues and hasa molecular weight of 3,483.

G-CSF may also be chosen as the active polypeptide Recombinantgranulocyte-colony stimulating factor or G-CSF is used following variouschemotherapy treatments to stimulate the recovery of white blood cells.

In one embodiment the biologically active polypeptide can be interferonalpha (IFN alpha). Chemically PEG-modified interferon-alpha 2a isclinically validated for the treatment of hepatitis C. In anotherembodiment the active polypeptide can be interferon gamma.

In one embodiment the biologically active polypeptide of a polypeptidefusion partner can be circularly permutated IL6. In a preferredembodiment, the biologically active polypeptide of a polypeptide fusionpartner is itself a fusion polypeptide comprising circularly permutatedIL-6 and the unpermutated D1 domain of gp130.

Additional cellular proteins include, but are not limited to: VEGF,VEGF-R1, VEGF-R2, VEGF-R3, Her-1, Her-2, Her-3, EGF-1, EGF-2, EGF-3,Alpha3, cMet, ICOS, CD40L, LFA-1, c-Met, ICOS, LFA-1, IL-6, B7.1, B7.2,OX40, IL-1b, TACI, IgE, BAFF, or BLys, TPO-R, CD19, CD20, CD22, CD33,CD28, IL-1-R1, TNFα, TRAIL-R1, Complement Receptor 1, FGFa, Osteopontin,Vitronectin, Ephrin A1-A5, Ephrin B1-B3, alpha-2-macroglobulin, CCL1,CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CXCL8, CXCL9, CXCL10, CXCL11,CXCL12, CCL13, CCL14, CCL15, CXCL16, CCL16, CCL17, CCL18, CCL19, CCL20,CCL21, CCL22, PDGF, TGFb, GMCSF, SCF, p40 (IL12/1L23), IL1b, IL1a,IL1ra, IL2, IL3, IL4, IL5, IL6, IL8, IL10, IL12, IL5, IL23, Fas, FasL,Fit3 ligand, 41BB, ACE, ACE-2, KGF, FGF-7, SCF, Netrin1,2, IFNa, b, g,Caspase-2, 3, 7, 8, 10, ADAM S1, S5, 8, 9, 15, TS1, TS5; Adiponectin,ALCAM, ALK-1, APRIL, Annexin V, Angiogenin, Amphiregulin,Angiopoietin-1, 2, 4, B7-1/CD80, B7-21CD86, B7-H1, B7-H2, B7-H3, Bcl-2.BACE-1, BAK, BCAM, BDNF, bNGF, bECGF, BMP2, 3, 4, 5, 6, 7, 8; CRP,Cadherin-6, 8, 11, Cathepsin A, B, C, D, E, L, S, V, X; CD11a/LFA-1,LFA-3, GP2b3a, GH receptor, RSV F protein, IL-23 (p40, p19), IL-12,CD80, CD86, CD28, CTLA-4, a4P1, a4137, TNF/Lymphotoxin, IgE, CD3, CD20,IL-6, IL-6R, BLYS/BAFF, IL-2R, HER2, EGFR, CD33, CD52, Digoxin, Rho (D),Varicella, Hepatitis, CMV, Tetanus, Vaccinia, Antivenom, Botulinum,Trail-R1, Trail-R2, cMet, TNF-R family, such as LA NGF-R, CD27, CD30,CD40, CD95, Lymphotoxin a/b receptor, Wsl-1, TL1A/TNFSF15, BAFF,BAFF-R/TNFRSF13C, TRAIL R2/TNFRSF10B, TRAIL R2/TNFRSF10B, Fas/TNFRSF6CD27/TNFRSF7, DR3/TNFRSF25, HVEM/TNFRSF14, TROY/TNFRSF19, CD40Ligand/TNFSF5, BCMA/TNFRSF17, CD30TNFRSF8, LIGHT/TNFSF14, 4-1BB/TNFRSF9, CD40/TNFRSF5, GITR/TNFRSF18, Osteoprotegerin/TNFRSF11B,RANK/TNFRSF11A, TRAIL R3/TNFRSF10C, TRAIL/TNFSF10, TRANCE/RANKL/TNFSF11, 4-1BB Ligand/TNFSF9, TWEAK/TNFSF12, CD40 Ligand/TNFSFS, FasLigand/TNFSF6, RELT/TNFRSF19L, APRIL/TNFSF13, DcR3/TNFRSF6B, TNFR1/TNFRSF1A, TRAIL R1/TNFRSF1A, TRAIL R4/TNFRSF10D, CD30 Ligand/TNFSF8,GITR Ligand/TNFSF18, TNFSF18, TACI/TNFRSF13B, NGF R/TNFRSF16, OX40Ligand/TNFSF4, TRAIL R2/TNFRSF10B, TRAIL R3/TNFRSF10C, TWEAK R/TNFRSF12,BAFF/BLyS/TNFSF13, DR6/TNFRSF21, TNF-alpha/TNFSF1A,Pro-TNF-alpha/TNFSF1A, Lymphotoxin beta R/TNFRSF3, Lymphotoxin beta R(LTbR)/Fc Chimera, TNF R1/TNFRSF1A, TNF-beta/TNFSF1B, PGRP-S, TNFR1/TNFRSF1A, TNF RII/TNFRSF1B, EDA-A2, TNF-alpha/TNFSF1A, EDAR, XEDAR,TNF RI/TNFRSF1A 4EBP1, 14-3-3 zeta, 53BP1, 2B4/SLAMF4, CCL21/6Ckine,4-1BB/TNFRSF9, 8D6A, 4-1BB Ligand/TNFSF9,8-oxo-dG,4-Amino-1,8-naphthalimide, A2B5, Aminopeptidase LRAP/ERAP2, A33,Aminopeptidase N/ANPEP, Aag, Aminopeptidase P2/XPNPEP2, ABCG2,Aminopeptidase P1/XPNPEP1, ACE, Aminopeptidase PILS/ARTS1, ACE-2,Amnionless, Actin, Amphiregulin, beta-Actin. AMPK alpha 1/2, Activin A,AMPK alpha 1, Activin AB, AMPK alpha 2, Activin B, AMPK beta 1, ActivinC, AMPK beta 2, Activin RIA/ALK-2, Androgen R/NR3C4, Activin RIB/ALK-4,Angiogenin, Activin RIIA, Angiopoietin-1, Activin RIIB, Angiopoietin-2,ADAMS, Angiopoietin-3, ADAM9, Angiopoietin-4, ADAM10, Angiopoietin-like1, ADAM12, Angiopoietin-like 2, ADAM15, Angiopoietin-like 3,TACE/ADAM17, Angiopoietin-like 4, ADAM19, Angiopoietin-like 7/CDT6,ADAM33, Angiostatin, ADAMTS4, Annexin A1/Annexin I, ADAMTSS, Annexin A7,ADAMTS1, Annexin A10, ADAMTSL-1/Punctin, Annexin V, Adiponectin/Acrp30,ANP, AEBSF, AP Site, Aggrecan, APAF-1, Agrin, APC, AgRP, APE, AGTR-2,APT, AIF, APLP-1, Akt, APLP-2, Akt1, Apolipoprotein AI, Akt2,Apolipoprotein B, Akt3, APP, Serum Albumin, APRIL/TNFSF13, ALCAM, ARC,ALK-1, Artemin, ALK-7, Arylsulfatase A/ARSA. Alkaline Phosphatase,ASAH2/N-acylsphingosine Amidohydrolase-2, alpha 2u-Globulin, ASC,alpha-1-Acid Glycoprotein, ASGR1, alpha-Fetoprotein, ASK1, ALS, ATM,Ameloblastin, ATRIP, AMICA/JAML, Aurora A, AMIGO, Aurora B, AMIGO2,Axin-1, AMIGO3, Ax1, Aminoacylase/ACY1, Azurocidin/CAP37/HBP,Aminopeptidase A/ENPEP, B4GALT1, BIM, B7-1/CD80, 6-Biotin-17-NAD,B7-2/CD86, BLAME/SLAMF8, B7-H1/PD-L1, CXCL13/BLC/BCA-1, B7-H2, BLIMP1,B7-H3, Blk, B7-H4, BMI-1, BACE-1, BMP-1/PCP, BACE-2, BMP-2, Bad, BMP-3,BAFF/TNFSF13B, BMP-3b/GDF-10, BAFF R/TNFRSF13C, BMP-4, Bag-1, BMP-5,BAK, BMP-6, BAMBI/NMA, BMP-7, BARD1, BMP-8, Bax, BMP-9, BCAM, BMP-10,Bcl-10, BMP-15/GDF-9B, Bcl-2, BMPR-IA/ALK-3, Bcl-2 related protein A1,BMPR-IB/ALK-6, Bcl-w, BMPR-II, Bcl-x, BNIP3L, Bcl-xL, BOC,BCMA/TNFRSF17, BOK, BDNF, BPDE, Benzamide, Brachyury, Common beta Chain,B-Raf, beta IG-H3, CXCL14/BRAK, Betacellulin, BRCA1, beta-Defensin 2,BRCA2, BID, BTLA, Biglycan, Bub-1, Bik-like Killer Protein, c-jun,CD90/Thy1, c-Rel, CD94, CCL6/C10, CD97, Clq R1/CD93, CD151, ClqTNF1,CD160, ClqTNF4, CD163, ClqTNF5, CD164, Complement Component C1r, CD200,Complement Component C1s, CD200 R1, Complement Component C2,CD229/SLAMF3, Complement Component C3a, CD23/Fc epsilon RII, ComplementComponent C3d, CD2F-10/SLAMF9, Complement Component CSa, CDSL,Cadherin-4/R-Cadherin, CD69, Cadherin-6, CDC2, Cadherin-8, CDC25A,Cadherin-11, CDC25B, Cadherin-12, CDCP1, Cadherin-13, CDO, Cadherin-17,CDX4, E-Cadherin, CEACAM-1/CD66a, N-Cadherin, CEACAM-6, P-Cadherin,Cerberus 1, VE-Cadherin, CFTR, Calbindin D, cGMP, Calcineurin A, ChemR23, Calcineurin B, Chemerin, Calreticulin-2, Chemokine Sampler Packs,CaM Kinase II, Chitinase 3-like 1, cAMP, Chitotriosidase/CHIT1,Cannabinoid R1, Chk1, Cannabinoid R2/CB2/CNR2, Chk2, CAR/NR113,CHL-1/L1CAM-2, Carbonic Anhydrase I, Choline Acetyltransferase/ChAT,Carbonic Anhydrase II, Chondrolectin, Carbonic Anhydrase III, Chordin,Carbonic Anhydrase IV, Chordin-Like 1, Carbonic Anhydrase Va,Chordin-Like 2, Carbonic Anhydrase VB, CINC-1, Carbonic Anhydrase VI,CINC-2, Carbonic Anhydrase VII, CINC-3. Carbonic Anhydrase VIII,Claspin, Carbonic Anhydrase IX, Claudin-6, Carbonic Anhydrase X, CLC,Carbonic Anhvdrase XII, CLEC-1, Carbonic Anhydrase XIII, CLEC-2,Carbonic Anhydrase XIV, CLECSF13/CLEC4F, Carboxymethyl Lysine, CLECSF8,Carboxypeptidase A1/CPA1, CLF-1, Carboxypeptidase A2, CL-P1/COLEC12,Carboxypeptidase A4, Clusterin, Carboxypeptidase B1, Clusterin-like 1,Carboxypeptidase E/CPE, CMG-2, Carboxypeptidase X1, CMV UL146,Cardiotrophin-1, CMV UL147. Carnosine Dipeptidase 1, CNP, Caronte, CNTF,CART, CNTF R alpha, Caspase, Coagulation Factor II/Thrombin, Caspase-1,Coagulation Factor III/Tissue Factor, Caspase-2, Coagulation Factor VII,Caspase-3, Coagulation Factor X, Caspase-4, Coagulation Factor XI,Caspase-6, Coagulation Factor XIV/Protein C, Caspase-7, COCO, Caspase-8,Cohesin, Caspase-9, Collagen I, Caspase-10, Collagen II, Caspase-12,Collagen IV, Caspase-13, Common gamma Chain/IL-2 R gamma, CaspasePeptide Inhibitors, COMP/Thrombospondin-5, Catalase, ComplementComponent C1rLP, beta-Catenin, Complement Component C1qA, Cathepsin 1,Complement Component C1qC, Cathepsin 3, Complement Factor D, Cathepsin6, Complement Factor I, Cathepsin A, Complement MASP3, Cathepsin B,Connexin 43, Cathepsin C/DPPL Contactin-1, Cathepsin D,Contactin-2/TAG1, Cathepsin E, Contactin-4, Cathepsin F, Contactin-5,Cathepsin H, Corin, Cathepsin L, Comulin, Cathepsin O, CORS26/C1qTNF, 3,Cathepsin S, Rat Cortical Stem Cells, Cathepsin V, Cortisol. CathepsinX/Z/P, COUP-TF I/NR2F1, CBP, COUP-TF II/NR2F2, CCI, COX-1, CCK-A R,COX-2, CCL28, CRACC/SLAMF7, CCR1, C-Reactive Protein, CCR2, CreatineKinase, Muscle/CKMM, CCR3, Creatinine, CCR4, CREB, CCR5, CREG, CCR6,CRELD1, CCR7, CRELD2, CCR8, CRHBP, CCR9, CRHR-1, CCR10, CRIM1,CD155/PVR, Cripto, CD2, CRISP-2, CD3, CRISP-3, CD4, Crossveinless-2,CD4+/45RA−, CRTAM, CD4+/45RO−, CRTH-2, CD4+/CD62L−/CD44, CRY1,CD4+/CD62L+/CD44, Cryptic, CD5, CSB/ERCC6, CD6, CCL27/CTACK, CD8.CTGF/CCN², CD8+/45RA−, CTLA-4, CD8+/45RO−, Cubilin, CD9, CX3CR1, CD14,CXADR, CD27/TNFRSF7, CXCL16, CD27 Ligand/TNFSF7, CXCR3, CD28, CXCR4,CD30/TNFRSF8, CXCR5, CD30 Ligand/TNFSF8, CXCR6, CD31/PECAM-1.Cyclophilin A, CD34, Cyr61/CCN1, CD36/SR-B3, Cystatin A, CD38, CystatinB, CD40/TNFRSF5, Cystatin C, CD40 Ligand/TNFSF5, Cystatin D, CD43,Cystatin E/M, CD44, Cystatin F, CD45, Cystatin H, CD46, Cystatin-2,CD47, Cystatin S, CD48/SLAMF2, Cystatin SA, CD55/DAF, Cystatin SN,CD58/LFA-3, Cytochrome c, CD59, Apocytochrome c, CD68, Holocytochrome c,CD72, Cytokeratin 8, CD74, Cytokeratin 14, CD83, Cytokeratin 19,CD84/SLAMF5, Cytonin, D6, DISP1, DAN, Dkk-1, DANCE, Dkk-2, DARPP-32,Dkk-3, DAX1/NROB1, Dkk-4, DCC, DLEC, DCIR/CLEC4A, DLL1, DCAR, DLL4,DcR3/TNFRSF6B, d-Luciferin, DC-SIGN, DNA Ligase IV, DC-SIGNR/CD299, DNAPolymerase beta, DcTRAIL R1/TNFRSF23, DNAM-1, DcTRAIL R2/TNFRSF22,DNA-PKcs, DDR1, DNER, DDR2, Dopa Decarboxylase/DDC, DEC-205, DPCR-1,Decapentaplegic, DPP6, Decorin, DPPA4, Dectin-1/CLEC7A, DPPA5/ESG1,Dectin-2/CLEC6A, DPPII/QPP/DPP7, DEP-1/CD148, DPPIV/CD26, DesertHedgehog, DR3/TNFRSF25, Desmin, DR6TNFRSF21, Desmoglein-1, DSCAM,Desmoglein-2, DSCAM-L1, Desmoglein-3, DSPG3, Dishevelled-1, Dtk,Dishevelled-3, Dynamin, EAR2/NR2F6, EphA5, ECE-1, EphA6, ECE-2, EphA7,ECF-L/CHI3L3, EphA8, ECM-1, EphB1, Ecotin, EphB2, EDA, EphB3, EDA-A2,EphB4, EDAR, EphB6, EDG-1, Ephrin, EDG-5, Ephrin-A1, EDG-8, Ephrin-A2,eEF-2, Ephrin-A3, EGF, Ephrin-A4, EGF R, Ephrin-A5, EGR1, Ephrin-B,EG-VEGF/PK1, Ephrin-B1, eIF2 alpha, Ephrin-B2, eIF4E, Ephrin-B3, Elk-1,Epigen, EMAP-II, Epimorphin/Syntaxin 2, EMMPRIN/CD147, Epiregulin,CXCL5/ENA, EPR-1/Xa Receptor, Endocan, ErbB2, Endoglin/CD105, ErB3,Endoglycan, ErbB4, Endonuclease III, ERCC1, Endonuclease IV, ERCC3,Endonuclease V, ERK1/ERK2, Endonuclease VIII, ERK1,Endorepellin/Perlecan, ERK2, Endostatin, ERK3, Endothelin-1, ERK5/BMK1,Engrailed-2, ERR alpha/NR3B1, EN-RAGE, ERR beta/NR3B2,Enteropeptidase/Enterokinase, ERR gamma/NR3B3, CCL11/Eotaxin,Erythropoietin, CCL24/Eotaxin-2, Erythropoietin R, CCL26/Eotaxin-3,ESAM, EpCAM/TROP-1, ER alpha/NR3A1, EPCR, ER beta/NR3A2, Eph,Exonuclease III, EphA1, Exostosin-like 2/EXTL2, EphA2, Exostosin-like3/EXTL3, EphA3, FABP1, FGF-BP, FABP2, FGF R1-4, FABP3, FGF R1, FABP4,FGF R2, FABP5, FGF R3, FABP7, FGF R4, FABP9, FGF R5, Complement FactorB, Fgr, FADD, FHR5, FAM3A, Fibronectin, FAM3B, Ficolin-2, FAM3C,Ficolin-3, FAM3D, FITC, Fibroblast Activation Protein alpha/FAP, FKBP38,Fas/TNFRSF6, Flap, Fas Ligand/TNFSF6, FLIP, FATP1, FLRG, FATP4, FLRT1,FATP5, FLRT2, Fc gamma R1/CD64, FLRT3, Fc gamma RIIB/CD32b, Flt-3, Fegamma RIIC/CD32c, Flt-3 Ligand, Fc gamma RIIA/CD32a, Follistatin, Fcgamma RIII/CD6, Follistatin-like 1, FcRH1/IRTA5, FosB/G0S3, FcRH2/IRTA4,FoxD3, FcRH4/IRTA1, FoxJ1, FcRH5/IRTA2, FoxP3, Fc Receptor-like3/CD16-2, Fpg, FEN-1, FPR1, Fetuin A, FPRL1, Fetuin B, FPRL2, FGFacidic, CX3CL1/Fractalkine, FGF basic, Frizzled-1, FGF-3, Frizzled-2,FGF-4, Frizzled-3, FGF-5, Frizzled-4, FGF-6, Frizzled-5, FGF-8,Frizzled-6, FGF-9, Frizzled-7, FGF-10, Frizzled-8, FGF-11, Frizzled-9,FGF-12, Frk, FGF-13, sFRP-1, FGF-16, sFRP-2, FGF-17, sFRP-3, FGF-19,sFRP-4, FGF-20, Furin, FGF-21, FXR/NR1H4, FGF-22, Fyn, FGF-23,G9a/EHMT2, GFR alpha-3/GDNF R alpha-3, GABA-A-R alpha 1, GFRalpha-4/GDNF R alpha-4, GABA-A-R alpha 2, GITR/TNFRSF18, GABA-A-R alpha4, GITR Ligand/TNFSF18, GABA-A-R alpha 5, GLI-1, GABA-A-R alpha 6,GLI-2, GABA-A-R beta 1, GLP/EHMT1, GABA-A-R beta 2, GLP-1 R, GABA-A-Rbeta 3, Glucagon, GABA-A-R gamma 2, Glucosamine(N-acetyl)-6-Sulfatase/GNS, GCABA-B-R2, GluR1, GAD1/GAD67, GluR2/3,GAD2/GAD65, GluR2, GADD45 alpha, GluR3, GADD45 beta, Glut1, GADD45gamma, Glut2, Galectin-1, Glut3, Galectin-2, Glut4, Galectin-3, GlutS,Galectin-3 BP, Glutaredoxin 1, Galectin-4, Glycine R, Galectin-7,Glycophorin A, Galectin-8, Glypican 2, Galectin-9, Glypican 3,GalNAc4S-65T, Glypican 5, GAP-43, Glypican 6, GAPDH, GM-CSF, Gas1,GM-CSF R alpha, Gas6, GMF-beta, GASP-1/WFIKKNRP, gp130, GASP-2/WFIKKN,Glycogen Phosphorylase BB/GPBB, GATA-1, GPR15, GATA-2, GPR39, GATA-3,GPVI, GATA-4, GR/N1R3C1, GATA-5, Gr-1/Ly-6G, GATA-6, Granulysin, GBL,Granzyme A, GCNFNR6A1, Granzyme B, CXCL6/GCP-2, Granzyme D, G-CSF,Granzyme G, G-CSF R, Granzyme H, GDF-1, GRASP, GDF-3 GRB2, GDF-5,Gremlin, GDF-6, GRO, GDF-7, CXCL1/GRO alpha, GDF-8, CXCL2/GRO beta,GDF-9, CXCL3/GRO gamma, GDF-11, Growth Hormone, GDF-15, Growth HormoneR, GDNF, GRP75/HSPA9B, GFAP, GSK-3 alpha/beta, GFI-1, GSK-3 alpha, GFRalpha-1/GDNF R alpha-1, GSK-3 beta, GFR alpha-2/GDNF R alpha-2, EGN1T,H2AX, Histidine, H60, HFM74A, HAI-1, HMGA2, HAI-2, HMGB1, HAI-2A,TCF-2/HNF-1 beta, HAI-2B, HNF-3 beta/FoxA2, HAND1, HNF-4 alpha/NR2A1,HAPLN1, HNF-4 gamma/NR2A2, Airway Trypsin-like Protease/HAT,HO-1/HMOX1/HSP32, HB-EGF, HO-2/HMOX2, CCL14a/HCC-1, HPRG, CCL14b/HCC-3,Hrk, CCL16/HCC-4, HRP-1, alpha HCG, HS6ST2, Hck, HSD-1, HCR/CRAM-A/B,HSD-2, HDGF, HSP10/EPF, Hemoglobin, HSP27, Hepassocin, HSP60, HIES-1,HSP70, HIES-4, HSP90, HGF, HTRA/Protease Do, HGF Activator,HTRA1/PRSS11, HGF R, HTRA2/Omi, HIF-1 alpha, HVEM/TNFRSF14, HIF-2 alpha,Hyaluronan, HIN-1/Secretoglobulin 3A1,4-Hydroxynonenal, Hip,CCL1/1-309/TCA-3, IL-10, cIAP (pan), IL-10 R alpha, cIAP-1/HIAP-2, IL-10R beta, cIAP-2/HIAP-1, IL-11, IBSP/Sialoprotein II, IL-11 R alpha,ICAM-1/CD54, 11-12, ICAM-2/CD102, IL-12/IL-23 p40, ICAM-3/CD50, IL-12 Rbeta 1, ICAM-5, IL-12 R beta 2, ICAT, IL-13, ICOS, IL-13 R alpha 1,Iduronate 2-Sulfatase/IDS, IL-13 R alpha 2, IFN, IL-15, IFN-alpha, IL-15R alpha, IFN-alpha 1, IL-16, IFN-alpha 2, IL-17, IFN-alpha 4b, IL-17 R,IFN-alpha A, IL-17 RCC, IFN-alpha B2, IL-17 RD, IFN-alpha C, IL-17B,IFN-alpha D, IL-17B R, IFN-alpha F, IL-17C, IFN-alpha G, IL-17D,IFN-alpha H2, IL-17E, IFN-alpha I, IL-17F, IFN-alpha J1, IL-18/IL-1F4,IFN-alpha K, IL-18 BPa, IFN-alpha WA, IL-18 BPc, IFN-alpha/beta R1,IL-18 BPd, IFN-alpha/beta R2, IL-18 R alpha/IL-1 R5, IFN-beta, IL-18 Rbeta/IL-1 R7, IFN-gamma, IL-19, IFN-gamma R1, IL-20, IFN-gamma R2, 11-20R alpha, IFN-omega, IL-20 R beta, IgE, IL-21, IGFBP-1, IL-21 R, IGFBP-2,IL-22, IGFBP-3, IL-22 R, IGFBP-4, IL-22BP, IGFBP-5, IL-23, IGFBP-6,IL-23 R, IGFBP-L1, IL-24, IGFBP-rp1/IGFBP-7, IL-26/AK155, IGFBP-rP10,IL-27, IGF-I, IL-28A, IGF-I R, IL-28B, IGF-II, IL-29/IFN-lambda 1,IGF-II R, IL-31, IgG, IL-31 RA, IgM, IL-32 alpha, IGSF2, L-33,IGSF4A/SynCAM, ILT2/CD85j, IGSF4B, ILT3/CD85k, IGSF8, ILT4/CD85d, IgY,ILT5/CD85a, IkB-beta, ILT6/CD85e, IKK alpha, Indian Hedgehog, IKKepsilon, INSRR, IKK gamma, Insulin, IL-1 alpha/IL-1F1, Insulin R/CD220,IL-1 beta/IL-1F2, Proinsulin, IL-1ra/IL-1F3, Insulysin/IDE, IL-F5/FIL1delta, Integrin alpha 2/CD49b, IL-1F6/1FIL1 epsilon, Integrin alpha3/CD49c, IL-1F7/FIL1 zeta, Integrin alpha 3 beta 1/VLA-3, IL-1F8/FIL1eta, Integrin alpha 4/CD49d, IL-1F9/IL-1H1, Integrin alpha 5/CD49e,IL-1F10/IL-1HY2, Integrin alpha 5 beta 1, IL-1 R1, Integrin alpha6/CD49f, IL-1 RII, Integrin alpha 7, IL-1 R3/IL-1 R AcP, Integrin alpha9, IL-1 R4/ST2, Integrin alpha E/CD103, IL-1 R6/IL-1 R rp2, Integrinalpha L/CD11a, IL-1 R8, Integrin alpha L beta 2, IL-1 R9, Integrin alphaM/CD11b, IL-2, Integrin alpha M beta 2, IL-2 R alpha, Integrin alphaV/CD51, L-2 R beta, Integrin alpha V beta 5, IL-3, Integrin alpha V beta3, IL-3 R alpha, Integrin alpha V beta 6, IL-3 R beta, Integrin alphaX/CD11c, IL-4, Integrin beta 1/CD29, IL-4 R, Integrin beta 2/CD18, IL-5,Integrin beta 3/CD61, IL-5 R alpha, Integrin beta 5, IL-6, Integrin beta6, IL-6 R, Integrin beta 7, IL-7, CXCL10/IP-10/CRG-2, IL-7 Ralpha/CD127, IRAK1, CXCR1/IL-8 RA, IRAK4, CXCR2/IL-8 RB, IRS-1,CXCL8/IL-8, Islet-1, IL-9, CXCL11/1-TAC, IL-9 R, Jagged 1, JAM-4/IGSFS,Jagged 2, JNK, JAM-A, JNK1/JNK2, JAM-B/VE-JAM, JNK1, JAM-C, JNK2,Kininogen, Kallikrein 3/PSA, Kininostatin, Kallikrein 4, KIR/CD158,Kallikrein 5, KIR2DL1, Kallikrein 6/Neurosin, KIR2DL3, Kallikrein 7,KIR2DL4/CD158d, Kallikrein 8/Neuropsin, KIR2DS4, Kallikrein 9, KIR3DL1,Plasma Kallikrein/KLKB1, KIR3DL2, Kallikrein 10, Kirrel2, Kallikrein 11,KLF4, Kallikrein 12, KLFS, Kallikrein 13, KLF6, Kallikrein 14, Klotho,Kallikrein 15, Klotho beta, KC, KOR, Keap1, Kremen-1, Kell, Kremen-2,KGF/FGF-7, LAG-3, LINGO-2, LAIR1, Lipin 2, LAIR2, Lipocalin-1, Lamininalpha 4, Lipocalin-2/NGAL, Laminin gamma 1,5-Lipoxygenase, Laminin I,LXR alpha/NR1H3, Laminin S, LXR beta/NR1H-12, Laminin-1, Livin,Laminin-5, LIX, LAMP, LMIR1/CD300A, Langerin, LMIR2CD300c, LAR,LMIR3/CD300LF, Latexin, LMIRS/CD300LB, Layilin, LMIR6/CD300LE, LBP,LMO2, LDL R, LOX-1/SR-E1, LECT2, LRH-1/NR5A2, LEDGF, LRIG1, Lefty,LRIG3, Lefty-1, LRP-1, Lefty-A, LRP-6, Legumain, LSECtin/CLEC4G, Leptin,Lumican, Leptin R, CXCL15/Lungkine, Leukotriene B4, XCL1/Lymphotactin,Leukotriene B4 R1, Lymphotoxin, LIF, Lymphotoxin beta/TNFSF3, LIF Ralpha, Lymphotoxin beta R/TNFRSF3, LIGHT/TNFSF14, Lyn, Limitin, Lyp,LIMPII/SR-B2, Lysyl Oxidase Homolog 2, LIN-28, LYVE-1, LINGO-1, alpha2-Macroglobulin, CXCL9/MIG, MAD2L1, Mimecan, MAdCAM-1, Mindin, MafB,Mineralocorticoid R/NR3C2, MafF, CCL3L1/MIP-1 alpha Isoform LD78 beta,MafG, CCL3/MIP-1 alpha, MafK, CCL4L1/LAG-1, MAG/Siglec-4-a, CCL4/MIP-1beta, MANF, CCL15/MIP-1 delta, MAP2, CCL9/10/MIP-1 gamma, MAPK, MIP-2,Marapsin/Pancreasin, CCL19/MIP-3 beta, MARCKS, CCL20/MIP-3 alpha, MARCO,MIP-1, Mash1, MIP-II, Matrilin-2, MIP-III, Matrilin-3, MIS/AMH,Matrilin-4, MIS R11, Matriptase/ST14, MIXL1, MBL, MKK3/MKK6, MBL-2,MKK3, Melanocortin 3R/MC3R, MKK4, MCAM/CD146, MKK6, MCK-2, MKK7, Mcl-1,MKP-3, MCP-6, MLH-1, CCL2/MCP-1, MLK4 alpha, MCP-1, MMP, CCL8/MCP-2,MMP-1, CCL7/MCP-3/MARC, MMP-2, CCL13/MCP-4, MMP-3, CCL12/MCP-5, MMP-7,M-CSF, MMP-8, M-CSF R, MMP-9, MCV-type II, MMP-10, MD-1, NMP-11, MD-2,MMP-11, MMP-12, CCL22/MDC, MMP-13, MDL-1/CLECSA, MMP-14, MDM2, MMP-15,MEA-1, MMP-16/MT3-MMP, MEK1/MEK2, MMP-24/MT5-MMP, MEK1, MMP-25/MT6-MMP,MEK2, MMP-26, Melusin, MMR, MEPE, MOG, Meprin alpha, CCL23/MPIF-1,Meprin beta, M-Ras/R-Ras3, Mer, Mrell, Mesothelin, MRP1 Meteorin,MSK1/MSK2, Methionine Aminopeptidase 1, MSK1, Methionine Aminopeptidase,MSK2, Methionine Aminopeptidase 2, MSP, MFG-E8, MSP R/Ron, MFRP, Mug,MgcRacGAP, MULT-1, MGL2, Musashi-1, MGMT, Musashi-2, MIA, MuSK, MICA,MutY DNA Glycosylase, MICB, MyD88, MICL/CLEC12A, Myeloperoxidase, beta 2Microglobulin, Myocardin, Midkine, Myocilin, MIF, Myoglobin, NAIP NGFI-Bgamma/NR4A3, Nanog, NgR2/NgRH1, CXCL7/NAP-2, NgR3/NgRH2, Nbs1,Nidogen-1/Entactin, NCAM-1/CD56, Nidogen-2, NCAM-L1, Nitric Oxide,Nectin-1, Nitrotyrosine, Nectin-2/CD112, NKG2A, Nectin-3, NKG2C,Nectin-4, NKG2D, Neogenin, NKp30, Neprilysin/CD10, NKp44,Neprilysin-2/MMEL1/MMEL2, NKp46/NCR1, Nestin, NKp80/KLRF1, NETO2,NKX2.5, Netrin-1, NMDA R, NR1 Subunit, Netrin-2, NMDA R, NR2A Subunit,Netrin-4, NMDA R, NR2B Subunit, Netrin-Gla, NMDA R, NR2C Subunit,Netrin-G2a, N-Me-6,7-diOH-TIQ, Neuregulin-1/NRG1, Nodal,Neuregulin-3/NRG3, Noggin, Neuritin, Nogo Receptor, NeuroD1, Nogo-A,Neurofascin, NOMO, Neurogenin-1, Nope, Neurogenin-2, Norrin,Neurogenin-3, eNOS, Neurolysin, iNOS, Neurophysin II, nNOS,Neuropilin-1, Notch-1, Neuropilin-2, Notch-2, Neuropoietin, Notch-3,Neurotrimin, Notch-4, Neurturin, NOV/CCN3, NFAM1, NRAGE, NF-H, NrCAM,NFkB1, NRL, NFkB2, NT-3, NF-L, NT-4, NF-M, NTB-A/SLAMF6, NG2/MCSP, NTH1,NGF R/TNFRSF16, Nucleostemin, beta-NGF, Nurr-1/NR4A2, NGFI-Balpha/NR4A1, OAS2, Orexin B, OBCAM, OSCAR, OCAM, OSF-2/Periostin,OCIL/CLEC2d, Oncostatin M/OSM, OCILRP2/CLEC21, OSM R beta, Oct-3/4,Osteoactivin/GPNMB, OGG1, Osteoadherin, Olig 1, 2, 3, Osteocalcin,Olig1, Osteocrin, Olig2, Osteopontin, Olig3, Osteoprotegerin/TNFRSF11B,Oligodendrocyte Marker 01, Otx2, Oligodendrocyte Marker 04, OV-6, OMgp,OX40, TNFRSF4, Opticin, OX40 Ligand/TNFSF4, Orexin A, OAS2, Orexin B,OBCAM, OSCAR, OCAM, OSF-2/Periostin, OCIL/CLEC2d, Oncostatin M/OSM,OCILRP2/CLEC21, OSM R beta, Oct-3/4, Osteoactivin/GPNMB, OGG1,Osteoadherin, Olig 1, 2, 3, Osteocalcin, Olig1, Osteocrin, Olig2,Osteopontin, Olig3, Osteoprotegerin/TNFRSF11B, Oligodendrocyte Marker01, Otx2, Oligodendrocyte Marker 04, OV-6, OMgp, OX40/TNFRSF4, Opticin,OX40 Ligand/TNFSF4, Orexin A, RACK1, Ret, Rad1, REV-ERB alphaNR1D1,Rad17, REV-ERB beta/NR1D2, Rad51, Rex-1, Rae-1, RGM-A, Rae-1 alpha,RGM-B, Rae-1 beta, RGM-C, Rae-1 delta, Rheb, Rae-1 epsilon, RibosomalProtein S6, Rae-1 gamma, RIP1, Raf-1, ROBO1, RAGE, ROBO2, Ra1A/Ra1B,ROBO3, Ra1A, ROBO4, Ra1B, ROR/NR1F1-3 (pan), RANK/TNFRSF11A, RORalpha/NR1F1, CCL5/RANTES, ROR gamma/NR1F3, Rap1A/B, RTK-like OrphanReceptor 1 ROR1, RAR alpha/NR1B1, RTK-like Orphan Receptor 2/ROR2, RARbeta/NR1B2, RP105, RAR gamma/NR1B3, RPA2, Ras, RSK (pan), RBP4,RSK1/RSK2, RECK, RSK1, Reg 2/PAP, RSK2, Reg I, RSK3, Reg II, RSK4, RegIII, R-Spondin 1, Reg Ma, R-Spondin 2, Reg IV, R-Spondin 3, Relaxin-1,RUNX1/CBFA2, Relaxin-2, RUNX2/CBFA1, Relaxin-3, RUNX3/CBFA3, RELM alpha,RXR alpha/NR2B1, RELM beta, RXR beta/NR2B2, RELT/TNFRSF19L, RXRgamma/NR2B3, Resistin, S100A10, SLITRK5, S100A8, SLP1, S100A9,SMAC/Diablo, S100B, Smad1, STOOP, Smad2, SALL1, Smad3,delta-Sarcoglycan, Smad4, Sca-1/Ly6, Smad5, SCD-1, Smad7, SCF, Smad8,SCF R/c-kit, SMC1, SCGF, alpha-Smooth Muscle Actin, SCL/Tall, SMUG1,SCP3/SYCP3, Snail, CXCL12/SDF-1, Sodium Calcium Exchanger 1,SDNSF/MCFD2, Soggy-1, alpha-Secretase, Sonic Hedgehog, gamma-Secretase,S or CS, beta-Secretase, S or CS3, E-Selectin, Sortilin, L-Selectin,SOST, P-Selectin, SOX1, Semaphorin 3A, SOX2, Semaphorin 3C, SOX3,Semaphorin 3E, SOX7, Semaphorin 3F, SOX9, Semaphorin 6A, SOX10,Semaphorin 6B, SOX17, Semaphorin 6C, SOX21 Semaphorin 6D, SPARC,Semaphorin 7A, SPARC-like 1, Separase, SP-D, Serine/ThreoninePhosphatase Substrate 1, Spinesin, Serpin A1, F-Spondin, Serpin A3,SR-AI/MSR, Serpin A4/Kallistatin, Src, Serpin A5/Protein C Inhibitor,SREC-I/SR-F1, Serpin A8/Angiotensinogen, SREC-II, Serpin B5, SSEA-1,Serpin C1/Antithrombin-III, SSEA-3, Serpin D1/Heparin Cofactor II,SSEA-4, Serpin E1/PAI-1, ST7/LRP12, Serpin E2, Stabilin-1, Serpin F1,Stabilin-2, Serpin F2, Stanniocalcin 1, Serpin G/C1 Inhibitor,Stanniocalcin 2, Serpin 12, STAT1, Serum Amyloid A1, STAT2, SF-1/NR5A,STAT3, SGK, STAT4, SHBG, STATSa/b, SHIP, STAT5a, SHP/NROB2, STAT5b,SHP-1, STAT6, SHP-2, VE-Statin, SIGIRR, Stella/Dppa3, Siglec-2/CD22,STRO-1, Siglec-3/CD33, Substance P, Siglec-5, Sulfamidase/SGSH,Siglec-6, Sulfatase Modifying Factor 1/SUMF1, Siglec-7, SulfataseModifying Factor 2/SUMF2, Siglec-9, SUMO1, Siglec-10, SUMO2/3/4,Siglec-11, SUMO3, Siglec-F, Superoxide Dismutase, SIGNR1/CD209,Superoxide Dismutase-1/Cu—Zn SOD, SIGNR4, Superoxide Dismutase-2/Mn-SOD,SIRP beta 1, Superoxide Dismutase-3/EC-SOD, SKI, Survivin, SLAM/CD150,Synapsin I, Sleeping Beauty Transposase, Syndecan-1/CD138, Slit3,Syndecan-2, SLITRK1, Syndecan-3, SLITRK2, Syndecan-4, SLITRK4,TACI/TNFRSF13B, TMEFF1/Tomoregulin-1, TAO2, TMEFF2, TAPP1,TNF-alpha/TNFSF1A, CCL17/TARC, TNF-beta/TNFSF1B, Tau, TNF R1/TNFRSF1A,TC21/R-Ras2, TNF R11/TNFRSF1B, TCAM-1, TOR, TCCR/WSX-1, TP-1, TC-PTP,TP63/TP73L, TDG, TR, CCL25/TECK, TR alpha/NR1A1, Tenascin C, TR beta1/NR1A2, Tenascin R, TR2/NR2C1, TER-119, TR4/NR2C2, TERT, TRA-1-85,Testican 1/SPOCK1, TRADD, Testican 2/SPOCK2, TRAF-1, Testican 3/SPOCK3,TRAF-2, TFPI, TRAF-3, TFPI-2, TRAF-4, TG F-alpha, TRAF-6, TGF-beta, TRAIL/TNFSF10, TGF-beta 1, TRAIL R1/TNFRSF10A, LAP (TGF-beta 1), TRAILR2/TNFRSF10B, Latent TGF-beta 1, TRAIL R3/TNFRSF10C, TGF-beta 1.2, TRAILR4/TNFRSF10D, TGF-beta 2, TRANCE/TNFSF11, TGF-beta 3, TfR (TransferrinR), TGF-beta 5, Apo-Transferrin, Latent TGF-beta bp1, Holo-Transferrin,Latent TGF-beta bp2, Trappin-2/Elafin, Latent TGF-beta bp4, TREM-1,TGF-beta RI/ALK-5, TREM-2, TGF-beta RII, TREM-3, TGF-beta RIIb,TREML1/TLT-1, TGF-beta RIII, TRF-1, Thermolysin, TRF-2, Thioredoxin-1,TRH-degrading Ectoenzyme/TRHDE, Thioredoxin-2, TRIMS, Thioredoxin-80,Tripeptidyl-Peptidase I, Thioredoxin-like 5/TRP14, TrkA, THOP1, TrkB,Thrombomodulin/CD141, TrkC, Thrombopoietin, TROP-2, Thrombopoietin R,Troponin I Peptide 3, Thrombospondin-1, Troponin T, Thrombospondin-2,TROY/TNFRSF19, Thrombospondin-4, Trypsin 1, Thymopoietin, Trypsin2/PRSS2, Thymus Chemokine-1, Trypsin 3/PRSS3, Tie-1, Tryptase-5/Prss32,Tie-2, Tryptase alpha/TPS1, TIM-1/KIM-1/HAVCR, Tryptase beta-1/MCPT-7,TLM-2, Tryptase beta-2/TPSB2, TIM-3, Tryptase epsilon/BSSP-4, TIM-4,Tryptase gamma-1/TPSG1, TIM-5, Tryptophan Hydroxylase, TIM-6, TSC22,TIMP-1, TSG, TIMP-2, TSG-6, TIMP-3, TSK, TIMP-4, TSLP, TL1A/TNFSF15,TSLP R, TLR1, TSP50, TLR2, beta-III Tubulin, TLR3, TWEAK/TNFSF12, TLR4,TWEAK R/TNFRSF12, TLRS, Tyk2, TLR6, Phospho-Tyrosine, TLR9, TyrosineHydroxylase, TLX/NR2E1, Tyrosine Phosphatase Substrate I, Ubiquitin,UNC5H3, Ugi, UNC5H4, UGRP1, UNG, ULBP-1, uPA, iULBP-2, uPAR, ULBP-3,URB, UNC5H1, UVDE, UNC5H2, Vanilloid R1, VEGF R, VASA, VEGF R1/Flt-1,Vasohibin, VEGF R2/KDR/Flk-1, Vasorin, VEGF R3/Flt-4, Vasostatin,Versican, Vav-1, VGSQ, VCAM-1, VI-HR, VDR/NR111, Vimentin, VEGF,Vitronectin, VEGF-B, VLDLR, VEGF-C, vWF-A2, VEGF-D, Synuclein-alpha,Ku70, WASP, Wnt-7b, WIF-1, Wnt-8a WISP-1/CCN4, Wnt-8b, WNK1, Wnt-9a,Wnt-1, Wnt-9b, Wnt-3a, Wnt-10a, Wnt-4, Wnt-10b, Wnt-5a, Wnt-11, Wnt-5b,wnvNS3, Wnt7a, XCR1, XPE/DDB1, XEDAR, XPE/DDB2, Xg, XPF, XIAP, XPG, XPA,XPV, XPD, XRCC1, Yes, YY1, EphA4.

Other active polypeptides include: BOTOX, Myobloc, Neurobloc, Dysport(or other serotypes of botulinum neurotoxins), alglucosidase alfa,daptomycin, YH-16, choriogonadotropin alfa, filgrastim, cetrorelix,interleukin-2, aldesleukin, teceleukin, denileukin diftitox, interferonalfa-n3 (injection), interferon alfa-n1, DL-8234, interferon, Suntory(gamma-1 a), interferon gamma, thymosin alpha 1, tasonermin, DigiFab,ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif,eptoterminalfa, teriparatide (osteoporosis), calcitonin injectable (bonedisease), calcitonin (nasal, osteoporosis), etanercept, hemoglobinglutamer 250 (bovine), drotrecogin alfa, collagenase, carperitide,recombinant human epidermal growth factor (topical gel, wound healing),DWP-401, darbepoetin alfa, epoetin omega, epoetin beta, epoetin alfa,desirudin, lepirudin, bivalirudin, nonacog alpha, Mononine, eptacog alfa(activated), recombinant Factor VIII+VWF, Recombinate, recombinantFactor VIII, Factor VIII (recombinant), Alphanate, octocog alfa, FactorVIIL palifermin, Indikinase, tenecteplase, alteplase, pamiteplase,reteplase, nateplase, monteplase, tfollitropin alfa, rFSH, hpFSH,micatfungin, pegfilgrastim, lenograstim, nartograstim, sermorelin,glucagon, exenatide, pramlintide, imiglucerase, galsulfase, Leucotropin,molgramostim, triptorelin acetate, histrelin (subcutaneous implant,Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained releasedepot (ATRIGEL), leuprolide implant (DUROS), goserelin, somatropin,Eutropin, KP-102 program, somatropin, somatropin, mecasermin (growthfailure), enfuvirtide, Org-33408, insulin glargine, insulin glulisine,insulin (inhaled), insulin lispro, insulin detemir, insulin (buccal,RapidMist), mecasermin rinfabate, anakinra, celmoleukin, 99 mTc-apcitideinjection, myelopid, Betaseron, glatiramer acetate, Gepon, sargramostim,oprelvekin, human leukocyte-derived alpha interferons, Bilive, insulin(recombinant), recombinant human insulin, insulin aspart, mecasermin,Roferon-A, interferon-alpha 2, Alfaferone, interferon alfacon-1,interferon alpha, Avonex′ recombinant human luteinizing hormone, dornasealfa, trafermin, ziconotide, taltirelin, diboterminalfa, atosiban,becaplermin, eptiibatide, Zemaira, CTC-111, Shanvac-B, H1-IPV vaccine(quadrivalent), NOV-002, octreotide, lanreotide, ancestim, agalsidasebeta, agalsidase alfa, laronidase, prezatide copper acetate (topicalgel), rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin,recombinant house dust mite allergy desensitization injection,recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis),epoetin delta, transgenic antithrombin 111, Granditropin, Vitrase,recombinant insulin, interferon-alpha (oral lozenge), GEM-21S,vapreotide, idursulfase, omapatrilat, recombinant serum albumin,certolizumab pegol, glucarpidase, human recombinant C1 esteraseinhibitor (angioedema), lanoteplase, recombinant human growth hormone,enfuvirtide (needle-free injection, Biojector 2000), VGV-1, interferon(alpha), lucinactant, aviptadil (inhaled, pulmonary disease), icatibant,ecallantide, omiganan, Aurograb, pexiganan acetate, ADI-PEG-20, LDI-200,degarelix, cintredekin besudotox, FavId, MDX-1379, ISAtx-247,liraglutide, teriparatide (osteoporosis), tifacogin, AA-4500, T4N5liposome lotion, catumaxomab, DWP-413, ART-123, Chrysalin, desmoteplase,amediplase, corifollitropin alpha, TH-9507, teduglutide, Diamyd,DWP-412, growth hormone (sustained release injection), recombinantG-CSF, insulin (inhaled, AIR), insulin (inhaled, Technosphere), insulin(inhaled, AERX), RGN-303, DiaPep277, interferon beta (hepatitis C viralinfection (HCV)), interferon alfa-n3 (oral), belatacept, transdermalinsulin patches, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX,GV-1001, LymphoScan, ranpimase, Lipoxysan, lusupultide, MP52(beta-tricalciumphosphate carrier, bone regeneration), melanoma vaccine,sipuleucel-T, CTP-37, Insegia, vitespen, human thrombin (frozen,surgical bleeding), thrombin, TransMID, alfimeprase, Puricase,terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinantFGF-1 (injectable, vascular disease), BDM-E, rotigaptide, ETC-216,P-113, MBI-594AN, duramycin (inhaled, cystic fibrosis), SCV-07, OPI-45,Endostatin, Angiostatin, ABT-510, Bowman Birk Inhibitor Concentrate,XMP-629, 99 mTc-Hynic-Annexin V, kahalalide F, CTCE-9908, teverelix(extended release), ozarelix, romidepsin, BAY-50-4798, interleukin-4,PRX-321, Pepscan, iboctadekin, rh lactoferrin, TRU-015, IL-21, ATN-161,cilengitide, Albuferon, Biphasix, IRX-2, omega interferon, PCK-3145,CAP-232, pasireotide, huN901-DM 1, ovarian cancer immunotherapeuticvaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16,multi-epitope peptide melanoma vaccine (MART-1, gp100, tyrosinase),nemifitide, rAAT (inhaled), rAAT (dermatological), CGRP (inhaled,asthma), pegsunercept, thymosin beta-4, plitidepsin, GTP-200,ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (oral, eligen),calcitonin (oral, osteoporosis), examorelin, capromorelin, Cardeva,velafermin, 131I-TM-601, KK-220, TP-10, ularitide, depelestat, hematide,Chrysalin (topical), rNAPc2, recombinant Factor VIII (PEGylatedliposomal), bFGF, PEGylated recombinant staphylokinase variant, V-10153,SonoLysis Prolyse, NeuroVax, CZEN-002, islet cell neogenesis therapy,rGLP-1, BIM-51077, LY-548806, exenatide (controlled release, Medisorb),AVE-0010, GA-GCB, avorelin, AOD-9604, linaclotide acetate, CETi-1,Hemospan, VAL (injectable), fast-acting insulin (injectable, Viadel),intranasal insulin, insulin (inhaled), insulin (oral, eligen),recombinant methionyl human leptin, pitrakinra subcutaneous injection,eczema), pitrakinra (inhaled dry powder, asthma), Multikine, RG-1068,MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmunediseases/inflammation), talactoferrin (topical), rEV-131 (ophthalmic),rEV-131 (respiratory disease), oral recombinant human insulin(diabetes), RPI-78M, oprelvekin (oral), CYT-99007 CTLA4-Ig, DTY-001,valategrast, interferon alfa-n3 (topical), IRX-3, RDP-58, Tauferon, bilesalt stimulated lipase, Merispase, alkaline phosphatase, EP-2104R,Melanotan-II, bremelanotide, ATL-104, recombinant human microplasmin,AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LABGHRH, AER-002, BGC-728, malaria vaccine (virosomes, PeviPRO), ALTU-135,parvovirus B 19 vaccine, influenza vaccine (recombinant neuraminidase),malaria, HBV vaccine, anthrax vaccine, Vacc-5q, Vacc-4×, HIV vaccine(oral), HPV vaccine, Tat Toxoid, YSPSL, CHS-13340, PTH(1-34) liposomalcream (Novasome), Ostabolin-C, PTH analog (topical, psoriasis),MBRI-93,02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine(tuberculosis), FAR-404, BA-210, recombinant plague F1V vaccine, AG-702,OXSODrol, rBetV1, Der-p1/Der-p2/Der-p7 allergen-targeting vaccine (dustmite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine,HPV-16 E7 lipopeptide vaccine, labyrinthin vaccine (adenocarcinoma), CMLvaccine, WTI-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin,CytoFab, P-9808, VT-111, icrocaptide, telbermin (dermatological,diabetic foot ulcer), rupintrivir, reticulose, rGRF, PIA,alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensintherapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral,tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin(anticancer), DT3881L-3, TST-10088, PRO-1762, Combotox,cholecystokinin-B/gastrin-receptor binding peptides, 111In-hEGF, AE-37,trastuzumab-DM 1, Antagonist G, IL-12 (recombinant), PM-02734, IMP-321,rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19 basedradioimmunotherapeutics (cancer), Re-188-P-2045, AMG-386, DC/1540/KLHvaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine(peptides), NA17.A2 peptides, melanoma vaccine (pulsed antigentherapeutic), prostate cancer vaccine, CBP-501, recombinant humanlactoferrin (dry eye), FX-06, AP-214, WAP-8294A2 (injectable), ACP-HIP,SUN-11031, peptide YY [3-36](obesity, intranasal), FGLL, atacicept,BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (nasal,osteoporosis), F-18-CCR1, AT-1001 (celiac disease/diabetes), JPD-003,PTH(7-34) liposomal cream (Novasome), duramycin (ophthalmic, dry eye),CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528,AMG-114, JR-013, Factor XIII, aminocandin, PN-951, 716155, SUN-E7001,TI-1-0318, BAY-73-7977, teverelix (immediate release), EP-51216, hGH(controlled release, Biosphere), OGP-I, sifuvirtide, TV-4710, ALG-889,Org-41259, rhCC10, F-991, thymopentin (pulmonary diseases), r(m)CRP,hepatoselective insulin, subalin, L19-IL-2 fusion protein, elafin,NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist(thrombocytopenic disorders), AL-108, AL-208, nerve growth factorantagonists (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide(eligen), GEM-OS 1, AC-162352, PRX-302, LFn-p24 fusion vaccine(Therapore), EP-1043, S. peumonmae pediatric vaccine, malaria vaccine,Neisserur meningutidis Group B vaccine, neonatal group B streptococcalvaccine, anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis mediatherapy, HCV vaccine (core antigen+ISCOMATRIX), hPTH(1-34) (transdermal,ViaDerm), 768974, SYN-101, PGN-0052, aviscumine, BIM-23190, tuberculosisvaccine, multi-epitope tyrosinase peptide, cancer vaccine, enkastim,APC-8024, G1-5005, ACC-001, TTS-CD3, vascular-targeted TNF (solidtumors), desmopressin (buccal controlled-release), onercept, TP-9201.

Of particular interest are known fusion proteins (linked to Fc domains,albumin, or transferrin, including bioactive peptides) comprising atherapeutic active protein that may be improved by adding (or replacingthe existing linker with) a mucin-domain polypeptide linker inaccordance to the invention including but not limited to, fusionproteins of: sTNFR2, CTLA4, TACI, LFA, IL-R, II-1RAcP, VEGF receptor,TPO receptor agonists, EPO receptor agonists, GLP-1, exendin-4.

The nucleic acid and amino acid sequences of numerous active proteinsare well known in the art and descriptions and sequences are availablein public databases such as Chemical Abstracts Services Databases (e.g.,the CAS Registry), GenBank, GenPept, Entrez Nucleotide, Entrez Protein,The Universal Protein Resource (UniProt) and subscription provideddatabases such as GenSeq (e.g., Derwent). Polynucleotide sequences maybe a wild type polynucleotide sequence encoding a given active protein(e.g., either full length or mature), or in some instances the sequencemay be a variant of the wild type polynucleotide sequence (e.g., apolynucleotide which encodes the wild type active protein, wherein theDNA sequence of the polynucleotide has been optimized, for example, forexpression in a particular species; or a polynucleotide encoding avariant of the wild type protein, such as a site directed mutant or anallelic variant. It is well within the ability of the skilled artisan touse a wild-type or consensus cDNA sequence or a codon-optimized variantof a active protein to create fusion protein constructs contemplated bythe invention using methods known in the art and/or in conjunction withthe guidance and methods provided herein, and described more fully inthe Examples.

Pharmacokinetic Properties of the Fusion Proteins

The invention provides fusion proteins of therapeutic active proteinswith enhanced pharmacokinetics compared to the therapeutic activeprotein not linked to a mucin-polypeptide domain, that, when used at theoptimal dose determined for the composition by the methods describedherein, can achieve enhanced pharmacokinetics compared to a comparabledose of the therapeutic active protein not linked to a mucin-domainpolypeptide in accordance with the invention. As used herein, a“comparable dose” means a dose with an equivalent moles/kg for thetherapeutic active protein that is administered to a subject in acomparable fashion. It will be understood in the art that a “comparabledosage” of the fusion protein would represent a greater weight of agentbut would have essentially the same mole-equivalents of the therapeuticactive protein in the dose of the fusion protein and/or would have thesame approximate molar concentration relative to the therapeutic activeprotein.

Pharmacokinetic properties that may be enhanced by using a mucin-domainpolypeptide linker in accordance with the invention include, but are notlimited to half-life, Tmax, Cmax (in this case, enhancement refers tothe reduction of peak-to-trough differences), distribution, or durationof action through a combination of the individual effects.

Physicochemical and Pharmaceutical Properties

In addition to enhancing the PK properties of a therapeutic, a fusionprotein comprising a mucin-domain polypeptide linker may useful forimproving the pharmaceutical or physicochemical properties (such as thedegree of aqueous solubility) of the therapeutic active peptide orprotein. Solubility improvements can be mediated both through additionof the highly hydrophilic carbohydrates on the mucin as well as throughselection of the proper mucin-polypeptide sequence, which mayadditionally contain ionizable residues such as aspartic acid, glutamicacid, histidine, lysine, and arginine. The ionizable residues result inthe modulation of the pI of the fusion protein and thereby the totalcharge of the protein in a formulation approaching physiological pH andtonicity.

The fusion proteins of the invention can be constructed and assayed,using methods described herein, to confirm the physicochemicalproperties of the fusion protein result in the desired properties. Inone embodiment, the mucin-domain polypeptide is selected such that thefusion protein has an aqueous solubility that is within at least about25% greater compared to a therapeutic active protein not linked to thefusion protein, or at least about 30%, or at least about 40%, or atleast about 50%, or at least about 75%, or at least about 100%, or atleast about 200%, or at least about 300%, or at least about 400%, or atleast about 500%, or at least about 1000% greater than the correspondingtherapeutic active protein not containing the mucin domain linker.

Uses of the Fusion Proteins

In another aspect, the invention provides a method of for achieving abeneficial effect in a disease, disorder or condition mediated bytherapeutic active protein. The present invention addresses certaindisadvantages and/or limitations of therapeutic active proteins whenfused to a polypeptide fusion partner in the absence of a mucin-domainpolypeptide linker.

In one embodiment, the invention provides a method for achieving abeneficial effect in a subject comprising the step of administering tothe subject a therapeutically or prophylactically-effective amount of afusion protein. The effective amount can produce a beneficial effect inhelping to treat a disease or disorder. In some cases, the method forachieving a beneficial effect can include administering atherapeutically effective amount of a fusion protein composition totreat a subject for diseases and disease categories wherein atherapeutic protein or peptide does not exist.

Diseases amenable to treatment by administration of the compositions ofthe invention include without limitation cancer, inflammatory diseases,arthritis, osteoporosis, infections in particular hepatitis, bacterialinfections, viral infections, genetic diseases, pulmonary diseases,diabetes, hormone-related disease, Alzheimer's disease, cardiacdiseases, myocardial infarction, deep vain thrombosis, diseases of thecirculatory system, hypertension, hypotension, allergies, pain relief,dwarfism and other growth disorders, intoxications, blot clottingdiseases, diseases of the innate immune system, embolism, wound healing,healing of burns, Crohn's disease, asthma, ulcer, sepsis, glaucoma,cerebrovascular ischemia, respiratory distress syndrome, corneal ulcers,renal disease, diabetic foot ulcer, anemia, factor IX deficiency, factorVIII deficiency, factor VII deficiency, mucositis, dysphagia,thrombocyte disorder, lung embolism, infertility, hypogonadism,leucopenia, neutropenia, endometriosis, Gaucher disease, obesity,lysosome storage disease, AIDS, premenstrual syndrome, Turners syndrome,cachexia, muscular dystrophy, Huntington's disease, colitis, SARS,Kaposi sarcoma, liver tumor, breast tumor, glioma, Non-Hodgkin lymphoma,Chronic myelocytic leukemia; Hairy cell leukemia; Renal cell carcinoma;Liver tumor; Lymphoma; Melanoma, multiple sclerosis, Kaposis sarcoma,papilloma virus, emphysema, bronchitis, periodontal disease, dementia,parturition, non-small cell lung cancer, pancreas tumor, prostate tumor,acromegaly, psoriasis, ovary tumor, Fabry disease, lysosome storagedisease.

In one embodiment, the method comprises administering a fusion proteinin accordance with the invention comprising to a mucin-domainpolypeptide linker and at least one pharmaceutically acceptable carrierto a subject in need thereof that results in greater improvement in atleast one parameter, physiologic condition, or clinical outcome mediatedby the fusion protein compared to the effect mediated by administrationof a pharmaceutical composition comprising a fusion protein in theabsence of a mucin-domain polypeptide linker administered at acomparable dose. In one embodiment, the pharmaceutical composition isadministered at a therapeutically effective dose. In another embodiment,the pharmaceutical composition is administered using multiplesimultaneous or sequential doses using a therapeutically effective doseregimen (as defined herein) for the length of the dosing period.

A therapeutically effective amount of a fusion protein may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of thefusion protein are outweighed by the therapeutically beneficial effects.A prophylactically effective amount refers to an amount of fusionprotein required for the period of time necessary to achieve the desiredprophylactic result.

In another aspect, the invention provides methods of making fusionproteins to result in increased stability, increased water solubility,and/or ease of formulation, as compared to the native therapeutic activeproteins. In one embodiment, the invention includes a method ofincreasing the aqueous solubility of a fusion protein as compared to afusion protein that does not comprise a mucin-domain polypeptide linker.Factors that contribute to the property of mucin-domain polypeptidelinker to confer increased water solubility on a fusion protein includethe high percentage of glycosylation, the type of glycans, and thecharge on the amino acids of the mucin-domain polypeptide. In someembodiments, the method results in a fusion protein wherein the watersolubility is at least about 50%, or at least about 60% greater, or atleast about 70% greater, or at least about 80% greater, or at leastabout 90% greater, or at least about 100% greater, or at least about150% greater, or at least about 200% greater, or at least about 400%greater, or at least about 600% greater, or at least about 800% greater,or at least about 1000% greater, or at least about 2000% greater, or atleast about 4000% greater, or at least about 6000% greater underphysiologic conditions, or in a therapeutically acceptable formulation,compared to the native therapeutic active protein.

Nucleic Acid Sequences

The present invention provides isolated polynucleic acids encodingfusion proteins and sequences complementary to polynucleic acidmolecules encoding fusion proteins of the invention. In another aspect,the invention encompasses methods to produce polynucleic acids encodingfusion proteins of the invention and sequences complementary to fusionproteins of the invention, including homologous variants. In general,the invention provides methods of producing a polynucleotide sequencecoding for a fusion protein and expressing the resulting gene productinclude assembling nucleotides encoding each of the mucin-domainpolypeptides and active proteins, linking the components in frame,incorporating the encoding gene into an appropriate expression vector,transforming an appropriate host cell with the expression vector, andcausing the fusion protein to be expressed in the transformed host cell,thereby producing the fusion protein of the invention. Standardrecombinant techniques in molecular biology can be used to make thepolynucleotides and expression vectors of the present invention. Inaccordance with the invention, nucleic acid sequences that encode afusion protein may be used to generate recombinant DNA molecules thatdirect the expression of fusion proteins in appropriate host cells.Several cloning strategies are envisioned to be suitable for performingthe present invention, many of which can be used to generate a constructthat comprises a gene coding for a fusion protein or its complement. Inone embodiment, the cloning strategy would be used to create a gene thatencodes a monomeric fusion protein that comprises an active protein anda mucin-domain polypeptide. In the foregoing embodiments hereinabovedescribed in this paragraph, the gene can further comprise nucleotidesencoding spacer sequences that may also encode cleavage sequence(s).

In one approach, a construct is first prepared containing the DNAsequence corresponding to a fusion protein. DNA encoding an activeprotein and/or a mucin polypeptide domain may be obtained from a cDNAlibrary prepared using standard methods from tissue or isolated cellsbelieved to possess the mRNA of an active protein and to express it at adetectable level. If necessary, the coding sequence can be obtainedusing conventional primer extension procedures as described in Sambrook,et al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA. Accordingly, DNAcan be conveniently obtained from a cDNA library prepared from suchsources. The encoding gene(s) may also be obtained from a genomiclibrary or created by standard synthetic procedures known in the art(e.g., automated nucleic acid synthesis) using DNA sequences obtainedfrom publicly available databases, patents, or literature references.Such procedures are well known in the art and well described in thescientific and patent literature. For example, sequences can be obtainedfrom Chemical Abstracts Services (CAS) Registry Numbers (published bythe American Chemical Society) and/or GenBank Accession Numbersavailable through the National Center for Biotechnology Information(NCBI) webpage, available on the world wide web at ncbi.nlm.nih.gov thatcorrespond to entries in the CAS Registry or GenBank database thatcontain an amino acid sequence of the active protein or of a fragment orvariant of the active protein or of the mucin-domain polypeptide.

A gene or polynucleotide encoding one or both of the polypeptide fusionpartners can be then be cloned into a construct, which can be a plasmidor other vector under control of appropriate transcription andtranslation sequences for high level protein expression in a biologicalsystem. In a later step, a second gene or polynucleotide coding for themucin-domain polypeptide linker, for example, is genetically fused tothe nucleotides encoding the N- and/or C-terminus of the polypeptidefusion partners by cloning it into the construct adjacent and in framenucleotides encoding the fusion partners.

The resulting polynucleotides encoding the fusion proteins can then beindividually cloned into an expression vector. The nucleic acid sequencemay be inserted into the vector by a variety of procedures. In general,DNA is inserted into an appropriate restriction endonuclease site(s)using techniques known in the art. Vector components generally include,but are not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan. Suchtechniques are well known in the art and well described in thescientific and patent literature.

Suitable vectors, hosts, and expression systems are well known to thoseskilled in the art of recombinant expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. Both expression and cloningvectors contain a nucleic acid sequence that enables the vector toreplicate in one or more selected host cells, and further allowsexpression and post-translational modification of the recombinantprotein within the host cell.

The present invention also provides a host cell for expressing themonomeric fusion protein compositions disclosed herein. Examples ofsuitable eukaryotic host cells include, but are not limited to yeasthosts such as Saccharomyces cerevisiae, Pichia pastoris, and Hansenulapolymorpha; insect hosts such as Spodoptera frupperda Sf9, Spodopterafrugiperda Sf21, and High Five cells; and mammalian hosts such as mousefibroblast cells (C 127-BPV), Chinese hamster ovary cells (CHO-DHFR,CHO-NEOSPLA, CHO-GS), and mouse myeloma cells (NSO-GS).

Expressed fusion proteins may be purified via methods known in the artor by methods disclosed herein. Procedures such as gel filtration,affinity purification, salt fractionation, ion exchange chromatography,size exclusion chromatography, hydroxyapatite adsorption chromatography,hydrophobic interaction chromatography and gel electrophoresis may beused; each tailored to recover and purify the fusion protein produced bythe respective host cells. Methods of purification are described inRobert K. Scopes, Protein Purification: Principles and Practice, CharlesR. Castor (ed.), Springer-Verlag 1994, and Sambrook, et al., supra.Multi-step purification separations are also described in Baron, et al.,Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J.Chromatogr. A. 679:67-83 (1994).

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprisingfusion proteins of the invention. In one embodiment, the pharmaceuticalcomposition comprises the fusion protein and at least onepharmaceutically acceptable carrier. Fusion proteins of the presentinvention can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the polypeptide iscombined with a pharmaceutically acceptable carrier vehicle, such asaqueous solutions or buffers, pharmaceutically acceptable suspensionsand emulsions. Examples of non-aqueous solvents include propyl ethyleneglycol, polyethylene glycol and vegetable oils. Therapeutic formulationsare prepared for storage by mixing the active ingredient having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers, as described in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the formof lyophilized formulations or aqueous solutions.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition, thepharmaceutical compositions can also contain other pharmaceuticallyactive compounds or a plurality of compounds of the invention.

More particularly, the present pharmaceutical compositions may beadministered for therapy by any suitable route including oral, rectal,nasal, topical (including transdermal, aerosol, buccal and sublingual),vaginal, parenteral (including subcutaneous, subcutaneous orintrathecally by infusion pump, intramuscular, intravenous andintradermal), intravitreal, and pulmonary. It will also be appreciatedthat the preferred route will vary with the therapeutic agent, conditionand age of the recipient, and the disease being treated.

In one preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

In one preferred embodiment, the pharmaceutical composition isadministered subcutaneously. In this embodiment, the composition may besupplied as a lyophilized powder to be reconstituted prior toadministration. The composition may also be supplied in a liquid form,which can be administered directly to a patient. In one embodiment, thecomposition is supplied as a liquid in a pre-filled syringe such that apatient can easily self-administer the composition.

In another embodiment, the compositions of the present invention areencapsulated in liposomes, which have demonstrated utility in deliveringbeneficial active agents in a controlled manner over prolonged periodsof time. Liposomes are closed bilayer membranes containing an entrappedaqueous volume. Liposomes may also be unilamellar vesicles possessing asingle membrane bilayer or multilamellar vesicles with multiple membranebilayers, each separated from the next by an aqueous layer. Thestructure of the resulting membrane bilayer is such that the hydrophobic(non-polar) tails of the lipid are oriented toward the center of thebilayer while the hydrophilic (polar) heads orient towards the aqueousphase. In one embodiment, the liposome may be coated with a flexiblewater soluble polymer that avoids uptake by the organs of themononuclear phagocyte system, primarily the liver and spleen. Suitablehydrophilic polymers for surrounding the liposomes include, withoutlimitation, PEG, polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose,polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences asdescribed in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973 and6,043,094, the contents of which are incorporated by reference in theirentirety.

Liposomes may be comprised of any lipid or lipid combination known inthe art. For example, the vesicle-forming lipids may benaturally-occurring or synthetic lipids, including phospholipids, suchas phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid,phosphatidylserine, phasphatidylglycerol, phosphatidylinositol, andsphingomyelin as disclosed in U.S. Pat. Nos. 6,056,973 and 5,874,104.The vesicle-forming lipids may also be glycolipids, cerebrosides, orcationic lipids, such as 1,2-dioleyloxy-3-(trimethylamino) propane(DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DORIE);N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3[N—(N′,N′-dimethylaminoethane) carbamoly] cholesterol (DC-Chol); ordimethyldioctadecylammonium (DDAB) also as disclosed in U.S. Pat. No.6,056,973. Cholesterol may also be present in the proper range to impartstability to the vesicle as disclosed in U.S. Pat. Nos. 5,916,588 and5,874,104.

For liquid formulations, a desired property is that the formulation besupplied in a form that can pass through a 25, 28, 30, 31, 32 gaugeneedle for intravenous, intramuscular, intraarticular, or subcutaneousadministration.

In other embodiments, the composition may be delivered via intranasal,buccal, or sublingual routes to the brain to enable transfer of theactive agents through the olfactory passages into the CNS and reducingthe systemic administration. Devices commonly used for this route ofadministration are included in U.S. Pat. No. 6,715,485. Compositionsdelivered via this route may enable increased CNS dosing or reducedtotal body burden reducing systemic toxicity risks associated withcertain drugs. Preparation of a pharmaceutical composition for deliveryin a subdermally implantable device can be performed using methods knownin the art, such as those described in, e.g., U.S. Pat. Nos. 3,992,518;5,660,848; and 5,756,115.

EXAMPLES

The following examples are offered by way of illustration and are not tobe construed as limiting the invention as claimed in any way.

Example 1 IL-1Ra-Mucin-Fc Fusion Protein Activity In Vitro

Fusion proteins of human IL-1Ra with the Fc domain from IgG1 were madewhere the IL-1Ra was either directly linked through the IgG1 hinge(RDB1800) (SEQ ID NO:1) or where 2 tandem repeats from human MUC20 wereinserted between the Fc domain and IL-1Ra (RDB1819) (SEQ ID NO:2). Thegenes were synthetically synthesized (Geneart) and cloned into pcDNA™(Invitrogen), then transiently expressed in CHO-S cells using FreeStyle™MAX Reagent (Life Technologies). Proteins were purified using Protein A(GE Healthcare) and dialyzed against PBS.

HEK-Blue™ IL-1β cells (InvivoGen) are human embryonic kidney cellsspecifically designed to detect bioactive IL-1β in vitro by monitoringthe IL-1β-induced activation of the NF-κB/AP-1 pathways. The cell lineexpresses an inducible secreted embryonic alkaline phosphatase (SEAP)reporter gene under control of the IFN-β minimal promoter fused to fiveMkt) and five AP-1 binding sites. For the IL-1β antagonist assay,HEK-Blue IL-1β cells were plated at 50,000 cells/well in DMEM mediacontaining 2 mM L-glu and 10% heat inactivated FBS (Gibco) and 57 pMIL-1β (R&D systems). Cells were incubated for 20 hours at 37° C., 5% CO₂with varying concentrations of IL-1Ra, RDB1800 or RDB1819. SEAPproduction was detected by adding QUANTI-Blue™ (InvivoGen) andincubating for 3 hours at 37° C., 5% CO₂ and then read on a plate readerat 630 nm.

IL-1β activation of the SEAP gene can be inhibited by the IL-1βantagonist IL-1Ra in a dose dependent manner. Loss of activity wasobserved for IL-1Ra when directly fused to Fc (RDB1800). Incorporationof the mucin domain between IL-1Ra and Fc partially restores theinhibitory activity of the IL-1Ra_Fc fusion molecule from an IC₅₀ of 98nM for 1800, to 23 nM for RDB1819 (FIG. 2).

Example 2 Molecular Weight Measurement

The addition of the mucin sequence is likely leading to a large increasein 0-glycosylation, and thus a larger hydrodynamic volume. RDB1800 andRDB1819 were characterized by analytical gel filtration on a Superdex200 10/300 GL column (GE Healthcare). The column was equilibrated at 0.5ml/min with PBS, pH 7.4 as a running buffer for all analyses. Afterequilibration, protein molecular weight standards (Gel Filtration HMWcalibration kit; GE Healthcare) were injected at a flow rate of 0.5ml/min in order to determine elution volume to generate a molecularweight standard curve. Purified samples of RDB1800 and RDB1819 were theninjected in separate runs at 0.5 mL/min to determine elution volume.Apparent molecular weights of RDB1800 and RDB1819 were determined byinterpolation using the standard curve generated from the elutionvolumes of the different molecular weight standards.

The apparent molecular weight of RDB1800 and RDB1819 was observed byanalytical size exclusion chromatography. There are a total of 4N-linked glycosylation sites present in RDB1800 and 1819, one in eachIL-Ra arm and one in each Fc portion of the molecule. The calculatedmolecular weight for RDB1800 is 85.4 kDa with an observed size of 117kDa. This observation is consistent with the presence of N-glycosylationand the flexible hinge connecting IL1Ra and Fc. The addition of themucin linker adds multiple O-linked glycosylation sites and a more rigidrod-like hinge region resulting in an observed size of 224 kDa comparedto a 95 kDa calculated molecular weight for RDB1819 (FIG. 3).

Example 3 IL-1Ra-Mucin-Fc Fusion Protein Activity In Vivo

Collagen antibody-induced arthritis (CAIA) is a mouse model ofrheumatoid arthritis (RA) that is known to involve the IL-1 pathway.Arthritis was stimulated on Day “−3” by administration of a cocktail ofmonoclonal antibodies that are directed at conserved auto-antigenicepitopes in collagen type II. At Day “0” an LPS endotoxin boost wasadministered, along with single subcutaneous (SC) injections of 20 mg/kgof RDB1800 or RDB1819. A group of ten mice were used for each treatment.Mouse paw volume displacement was measured across multiple days toassess the degree of paw inflammation.

RDB1800 and RDB1819 significantly reduced paw edema for up to 10 daysand 14 days after injection respectively, as compared to the Salinecontrol group. Additionally, the increased potency observed in the invitro HEK bioassay correlates with an increase in potency in the in vivoCAIA model with RDB1819 showing a statistically significant differencein paw reduction compared to RDB1800 (FIG. 4).

Example 4 Inhibition of IL-6-Dependent Differentiation of M1 Cells byGp130-Mucin-Fc Fusion Construct

Fusion proteins comprising a truncated human gp130 comprising domainsD1-D3 and the Fc domain from IgG1 were made +/−mucin domains (RDB1601,SEQ ID NO:3 and RDB1613 SEQ ID NO: 4). The genes were syntheticallysynthesized (Geneart) and cloned into pcDNA™ (Invitrogen) andtransiently expressed in CHO-S cells using FreeStyle™ MAX Reagent (LifeTechnologies). Proteins were purified using Protein A (GE Healthcare)and dialyzed against PBS.

In vitro bioactivity was assessed by evaluating the ability of RDB1601and RDB1613 to inhibit IL-6-dependent differentiation of M1 cells, asmeasured by the percent of CD32 positive cells after exposure to IL-6.For the M1 assay, 75,000 were stimulated with 4 ng/mL IL-6 and 125 ng/mLIL-6Ra in 1:1 mix of DMEM/MEM media containing 10% FBS, 2×NEAA, 2×vitamin solution (Gibco). Inhibition was tested by the addition ofvarying concentrations of RDB1601 and RDB1613 and incubated for 72 hoursat 37° C., 5% CO₂. M1 cells were then stained with an anti-mouse CD32-PEantibody (R&D Systems) and analyzed by flow cytometry.

RDB1601 (non-mucin linker construct) inhibited IL-6-dependentdifferentiation of M1 cells in a dose dependent fashion with an IC₅₀ of20.1 nM, whereas the mucin-containing construct RDB1613 had an IC₅₀ of2.5 nM. (FIG. 5). Thus, RDB1613 is eight-fold more potent than thenon-mucin linker construct RDB1601.

Example 5 Inhibition of IL-6-Dependent Differentiation of M1 Cells bycpIL-6-gp130D1-Mucin-(IgG2)Fc Fusion Protein RDB1562 (SEQ ID NO:19)

Fusion proteins comprising a circularly permuted IL-6 (cpIL-6), the D1domain of human gp130 (gp130D1) and the Fc domain from IgG2 wereexpressed with (RDB1562, SEQ ID NO:19) and without (RDB1542, SEQ ID NO:18) 2 tandem repeats from human MUC20 inserted between the gp130D1domain and the Fc domain. The genes were synthesized (Geneart) andcloned into pcDNA™ (Invitrogen) and transiently expressed in CHO-S cellsusing FreeStyle™ MAX Reagent (Life Technologies). Proteins were purifiedusing Protein A (GE Healthcare) and dialyzed against PBS. In vitrobioactivity was assessed by evaluating the ability of RDB1542 andRDB1562 to inhibit IL-6-dependent differentiation of M1 cells, asmeasured by the percent of CD32 positive cells after exposure to IL-6,as described in Example 4.

Both RDB1542 (the non-mucin linker construct) and RDB1562 (themucin-containing construct) inhibited IL-6-dependent differentiation ofM1 cells in a dose dependent fashion with IC₅₀s of >1.0 mM and 2.6 nM,respectively (FIG. 6). Thus, insertion of the mucin domain as a linkerresults in a molecule that is >400-fold more potent than the equivalentmolecule without the mucin linker.

Example 6 Inhibition of IL-1β-Dependent Signaling byIL-1Ra-Mucin-(IgG2)Fc Fusion Protein RDB1840 (SEQ ID NO:21)

Fusion proteins of human IL-1Ra with the Fc domain from IgG2 were madewhere the IL-1Ra was either directly linked through the IgG2 hinge(RDB1841, SEQ ID NO:20) or where 2 tandem repeats from human MUC20 wereinserted between the Fc domain and IL-1Ra (RDB1840, SEQ ID NO:21). Thegenes were synthesized (Geneart) and cloned into pcDNA™ (Invitrogen) andtransiently expressed in CHO-S cells using FreeStyle™ MAX Reagent (LifeTechnologies). Proteins were purified using Protein A (GE Healthcare)and dialyzed against PBS. In vitro bioactivity was assessed byevaluating the ability of RDB1840 and RDB1841 to inhibit IL-1β-dependentsignaling in HEK-Blue™ IL-1β cells (InvivoGen), as described in Example1.

Both RDB1841 and RDB1840 inhibited IL-1β-dependent signaling in a dosedependent fashion. RDB1840, containing the mucin linker, inhibitedsignaling with about 12-fold greater potency (IC₅₀ of 5.0 nM) thanRDB1841, lacking the mucin linker, (IC₅₀ of 63.0 nM) (FIG. 7).

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It should also be understood thatthe embodiments described herein are not mutually exclusive and thatfeatures from the various embodiments may be combined in whole or inpart in accordance with the invention.

SEQUENCE LISTING 1) PRT >RDB1800 (SEQ. ID. NO. 1) 1MDAMKRGLCCVLLLCGAVFVSARRPSGRKSSKMQAFRIWDVNQKTFYLR NNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDK RFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDETGGGTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 2) PRT >RDB1819(SEQ. ID. NO. 2) 1 MDAMKRGLCCVLLLCGAVFVSARRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKID VVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACP GWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDESGSGGASSESSASSDGPHPVITESRASSESSASSD GPHPVITESREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3) PRT >RDB1601(SEQ. ID. NO. 3) 1 MDAMKRGLCCVLLLCGAVFVSARLLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITTGGGPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 4) PRT >RDB1613(SEQ. ID. NO. 4) 1 MYRMQLLSCIALSLALVTNSLLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRGGGSGGGASSESSASSDGPHPVITESRASSESSASSDGPHPVITESREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 5) PRTMUC1 tandem repeat unit (multiple variants) 1 PAPGSTAPPA HGVTSAPDTR 6)PRT MUC2 tandem repeat unit 1 ITTTTTVTPT PTPTGTQTPT TTP 7) PRT MUC3(A)tandem repeat unit (degenerate sequence) 1 ITTTETTSHD TPSFTSS 8) PRTMUC4 tandem repeat unit (degenerate sequence) 1 ATPLPVTDTS SASTGH 9) PRTMUC5AC tandem repeat unit 1 TTSTTSAP 10) PRT MUC5B tandem repeat unit(degenerate sequence) 1 ATGSTATPSS TPGTTHTPPV LTTTATTPT 11) PRT MUC7tandem repeat unit (degenerate sequence) 1 TTAAPPTPSA TTQAPPSSSA PPE 12)PRT MUC11/12 tandem repeat unit 1 EESTTVHSSP GATGTALFP 13) PRT MUC17tandem repeat unit (degenerate sequence) 1 SSSPTPAEGT SMPTSTYSEGRTPLTSMPVS TTLVATSAI STLSTTPVDT STPVTNSTEA 14) PRT MUC20 tandem repeatunit 1 SESSASSDGP HPVITPSRA 15) PRT MUC21 tandem repeat unit (degeneratesequence) 1 ATNSESSTVS SGIST 16) PRT TIM-1 tandem repeat unit(degenerate sequence) 1 VPTTTT 17) PRT PSGL-1 tandem repeat unit(degenerate sequence) 1 QTTQPAATEA 18) PRT RDB1542 1QNQWLQDMTTHLILRSFKEFLQSSLRALRQMSGGSGGGSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQASELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGGSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 19) PRTRDB1562 1 QNQWLQDMTTHLILRSFKEFLQSSLRALRQMSGGSGGGSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQASELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGGGGGSASSESSASSDGPHPVITESRASSESSASSDGPHPVITESRSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 20) PRTRDB1841 1 RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 21) PRT RDB1840 1RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDESGSGGASSESSASSDGPHPVITESRASSESSASSDGPHPVITESRERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1-20. (canceled)
 21. A fusion protein comprising a first polypeptidefusion partner and a second polypeptide fusion partner wherein the firstfusion partner is linked to the second fusion partner by a mucin-domainpolypeptide linker, wherein the first fusion partner is: sTNFR2, CTLA4,TACI, LFA, IL-10, IL-1RI, IL-1RAcP, VEGF receptor, TPO receptoragonists, EPO receptor agonists, GLP-1, exendin-4 or an amino acidsequence homologous to any of the foregoing with at least 90% sequenceidentity at the amino acid level, and wherein the second fusionpolypeptide comprises all or a portion of an immunoglobulin comprisingan Fc region, and wherein the mucin domain polypeptide linker comprisesat least one tandem repeat comprising SEQ ID NO:
 14. 22. The fusionprotein of claim 21, wherein the mucin-domain polypeptide linkercomprises between 1 and 5 domains of tandem repeats.
 23. The fusionprotein of claim 21, comprising a mucin-domain polypeptide linker of10-100 total residues.
 24. A composition comprising the fusion proteinof claim 21, and at least one pharmaceutically acceptable carrier.
 25. Afusion protein of claim 21, wherein the first fusion partner is IL-1RIor an amino acid sequence homologous to any of the foregoing with atleast 90% sequence identity at the amino acid level.
 26. The fusionprotein of claim 25, having the amino acid sequence of SEQ ID NO: 2 oran amino acid sequence homologous to any of the foregoing with at least90% sequence identity at the amino acid level.